Oligonucleotide analogues incorporating 5-aza-cytosine therein

ABSTRACT

Oligonucleotide analogs are provided that incorporate 5-aza-cytosine in the oligonucleotide sequence, e.g., in the form of 5-aza-2′-deoxycytidine (decitabine) or 5-aza-cytidine. In particular, oligonucleotide analogs rich in decitabine-deoxyguanosine islets (DpG and GpD) are provided to target the CpG islets in the human genome, especially in the promoter regions of genes susceptible to aberrant hypermethylation. Such analogs can be used for modulation of DNA methylation, such as effective inhibition of methylation of cytosine at the C-5 position. Methods for synthesizing these oligonucleotide analogs and for modulating nucleic acid methylation are provided. Also provided are phosphoramidite building blocks for synthesizing the oligonucleotide analogs, methods for synthesizing, formulating and administering these compounds or compositions to treat conditions, such as cancer and hematological disorders.

PRIORITY

This application is a continuation of U.S. application Ser. No.13/894,288, filed May 14, 2013, which is a continuation of U.S.application Ser. No. 12/703,096, filed Feb. 9, 2010, now U.S. Pat. No.8,461,123, which is a continuation of U.S. application Ser. No.11/241,799, filed Sep. 29, 2005, now U.S. Pat. No. 7,700,567, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Dec. 22, 2015 is named12636350303.txt and is 12,531 bytes in size.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to design, synthesis and application ofoligonucleotide analogues which are useful as therapeutics, diagnosticsas well as research reagents. Oligonucleotide analogues are providedthat incorporate an analogue of cytosine, 5-aza-cytosine, in theoligonucleotide sequence, e.g., in the form of 5-aza-2′-deoxycytidine or5-aza-cytidine. Such analogues can be used for modulation of DNAmethylation, especially for effective inhibition of methylation ofcytosine at the C-5 position by more specifically targeting the CpGislets of the human genome. Methods for synthesizing theseoligonucleotide analogues and for modulating C-5 cytosine methylationare provided. In particular, phosphoramidite building blocks andoligonucleotides containing decitabine (5-aza-2′-deoxycytidine; D),DpG-rich (Decitabine-phosphodiester linkage-Guanosine) islets andderivatives, are provided. Also provided are methods for preparing,formulating and administering these compounds or compositions astherapeutics to a host in need thereof.

2. Description of Related Art

Decitabine is currently being developed as a new pharmaceutical for thetreatment of chronic myelogenous leukemia (CML), myelodysplasticsyndrome (MDS), non-small cell lung (NSCL) cancer, sickle-cell anemia,and acute myelogenous leukemia (AML). Two isomeric forms of decitabinecan be distinguished. The β-anomer is the active form, which is shown inFIG. 1. Decitabine possesses multiple pharmacological characteristics.At the molecular level, it is incorporated into DNA during the S phaseof cell cycle. At the cellular level, decitabine can induce celldifferentiation and exert hematological toxicity. Despite having a shorthalf-life in vivo, decitabine has an excellent tissue distribution.

One of the functions of decitabine is its ability to specifically andpotently inhibit DNA methylation. DNA methylation is an epigeneticeffect common to many systems. This modification involves the covalentmodification of cytosine at the C-5 position (1a″). Methylation patternsare stably maintained at CpG dinucleotides by a family of DNAmethyltransferases that recognize hemimethylated DNA after DNAreplication. Inside the cell, decitabine is first converted into itsactive form, the phosphorylated 5-aza-deoxycytidine, by deoxycytidinekinase, which is primarily synthesized during the S phase of the cellcycle. The affinity of decitabine for the catalytical site ofdeoxycytidine kinase is similar to the natural substrate, deoxycytidine(Momparler et al. 1985 Mol. Pharmacol. 30:287-299). After conversion toits triphosphate form by deoxycytidine kinase, decitabine isincorporated into replicating DNA at a rate similar to that of thenatural substrate, dCTP (Bouchard and Momparler 1983 Mol. Pharmacol.24:109-114).

CpG-rich sequences of housekeeping genes are generally protected frommethylation in normal cells. In cancerous cells, aberranthypermethylation in promoter region CpG-islands of tumor suppressorgenes is one of the most common events associated with progression ofthe tumorigenic phenotype. Each class of differentiated cells has itsown distinct methylation pattern. Incorporation of decitabine into theDNA strand has a hypomethylation effect. After chromosomal duplication,in order to conserve this pattern of methylation, the 5-methylcytosineon the parental strand serves to direct methylation on the complementarydaughter DNA strand. Substituting the carbon at the C-5 position of thecytosine for nitrogen interferes with this normal process of DNAmethylation. The replacement of cytosine with decitabine at a specificsite of methylation produces an irreversible inactivation of DNAmethyltransferases. Decitabine behaves faithfully as a cytosine residueuntil DNA methyltransferase enzymes attempt to transfer a methyl groupto the hemimethylated DNA strands of the daughter cells. At this stepthe DNA methyltransferase enzyme is covalently trapped by decitabine inthe DNA and cannot further silence (methylate) additional cytosineresidues (Juttermann et al. 1994 Proc. Natl. Acad. Sci. USA91:11797-11801). This unique mechanism of action of decitabine allowsgenes silenced (that were once methylated) from previous rounds of celldivision to be re-expressed. The active trap is present in thehemimethylated DNA up to 48 hours after decitabine treatment. Afterfurther DNA synthesis and cell cycle division, progeny strands from thehemimethylated DNA result in DNA strands that are completelyunmethylated at these sites (Jones P. 2001 Nature 409: 141, 143-4). Byspecifically inhibiting DNA methyltransferases, the enzyme required formethylation, aberrant methylation of the tumor suppressor genes could bereversed.

Despite its proven antileukemic effects in CML, MDS, and AML, thepotential application of decitabine has been hampered by delayed andprolonged myelosuppression. Lower doses of decitabine, given over alonger period of time, have minimized myelosuppression to manageablelevels without compromising its ability to suppress cancer via itshypomethylation effect. At higher doses, the associated toxicity wasprohibitive. However, treatment of hematologic and solid tumors atmaximally tolerated doses of decitabine has been ineffective. The causeof myelosuppression is not clear. It is plausible that since decitabineis randomly and extensively incorporated into the DNA of S phase cells,including bone marrow cells that are involved in normal hematopoiesis,the severe DNA damage due to the instability of decitabine leads tonecrosis. Since incorporation of decitabine is not restricted to onlythe CpG-rich sequences, the DNA can break, due to the instability ofdecitabine, and require repair at numerous sites outside of the CpGislands.

Decitabine and azacitidine are unstable in aqueous media and undergohydrolytic degradation. In acidic medium, decitabine is hydrolyzed atroom temperature to 5-azacytosine and 2-deoxyribose. In neutral mediumat room temperature, the opening of the triazine ring takes place at the6-position to form the transient intermediate formyl derivative, whichfurther degrades to the amidino-urea derivative and formic acid(Piskala, A.; Synackova, M.; Tomankova, H.; Fiedler, P.; Zizkowsky, V.Nucleic Acids Res. 1978, 4, s109-s-113). This hydrolysis at the6-position occurs in acidic and basic aqueous media at even fasterrates.

In view of the chemical instability and toxicities associated withdecitabine, there exists a need to develop not only more stablederivatives of decitabine but superior hypomethylating agents, whereincorporation is localized to the CpG islands as much as possible orhypomethylation is achieved without significantly affecting theintegrity of the DNA.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotide analogues thatincorporate 5-aza-cytosine in the oligonucleotide sequence, e.g., in theform of 5-aza-2′-deoxycytidine (decitabine) or 5-aza-cytidine.

In one aspect of the invention, an isolated or synthetic oligonucleotideanalogue having 12 or less bases in length is provided, which comprisesone or more 5-aza-cytosine residues in the sequence of theoligonucleotide analogue.

In an embodiment, the oligonucleotide analogue has a general formula:—Z-L-G-, or -G-L-Z—,wherein Z is 5-aza-cytosine; G is guanine; and L is a chemical linkercovalently linking Z and G. The oligonucleotide analogue optionally hasmore than 30%, 35%, or 40% guanine residues in the sequence of theoligonucleotide analogue.

In particular embodiments, the oligonucleotide analogue is selected fromthe group consisting of 5′-DpG-3′, 5′-GpD-3′, 5′-DpGpD-3′, 5′-GpGpD-3′,5′-GpDpG-3′, 5′-GpDpD-3′, 5′-DpDpG-3′, 5′-DpGpG-3′, 5′-GpDpD-3′,5′-DpGpA-3′, 5′-DpGpDpG-3′, 5′-DpGpGpD-3′, 5′-GpDpGpD-3′, 5′-GpDpDpG-3′,5′-DpGpDpGpA-3′, wherein D is decitabine; p is a phospholinker; A is2′-deoxyadenosine, and G is 2′-deoxyguanosine.

In another aspect of the invention, an isolated or syntheticoligonucleotide analogue is provided which comprises, 2 or more copiesof a dinucleotide analogue having the general formula:—Z-L-G-, or -G-L-Z—,wherein Z is 5-aza-cytosine; G is guanine; and L is a chemical linkercovalently linking Z and G.

Optionally, the oligonucleotide analogue comprises less than 10, 8, 6,or 4 copies of the dinucleotide analogue —Z-L-G-, or -G-L-Z—.

In particular embodiments, the oligonucleotide analogue comprises asegment selected from the group consisting of 5′-DpG-3′, 5′-GpD-3′,5′-DpGpD-3′, 5′-GpGpD-3′, 5′-GpDpG-3′, 5′-GpDpD-3′, 5′-DpDpG-3′,5′-DpGpG-3′, 5′-GpDpD-3′, 5′-DpGpA-3′, 5′-DpGpDpG-3′, 5′-DpGpGpD-3′,5′-GpDpGpD-3′, 5′-GpDpDpG-3′, 5′-DpGpDpGpA-3′, wherein D is decitabine;p is a phospholinker; A is 2′-deoxyadenosine, and G is2′-deoxyguanosine.

In yet another aspect of the invention, an isolated or syntheticoligonucleotide analogue having at least 6 bases in length is provided,which comprises one or more 5-aza-cytosine residues in the sequence ofthe oligonucleotide analogue and has at least 75% sequence homology witha segment of a gene, preferably the 5′-untranslated region of a gene,such as the promoter of the gene.

In an embodiment, the oligonucleotide analogue has a general formula:—Z-L-G-, or -G-L-Z—,wherein Z is 5-aza-cytosine; G is guanine; and L is a chemical linkercovalently linking Z and G. The oligonucleotide analogue optionally hasmore than 30%, 35%, or 40% guanine residues in the sequence of theoligonucleotide analogue.

In particular embodiments, the oligonucleotide analogue comprises asegment selected from the group consisting of 5′-DpG-3′, 5′-GpD-3′,5′-DpGpD-3′, 5′-GpGpD-3′, 5′-GpDpG-3′, 5′-GpDpD-3′, 5′-DpDpG-3′,5′-DpGpG-3′, 5′-GpDpD-3′, 5′-DpGpA-3′, 5′-DpGpDpG-3′, 5′-DpGpGpD-3′,5′-GpDpGpD-3′, 5′-GpDpDpG-3′, 5′-DpGpDpGpA-3′, wherein D is decitabine;p is a phospholinker; A is 2′-deoxyadenosine, and G is2′-deoxyguanosine.

In yet another aspect of the invention, an oligonucleotide analogue isprovided that binds an allosteric site on DNA methyltransferases therebyinhibiting DNA methyltransferases.

In one embodiment, the oligonucleotide analogue has a sequence of

5′-CTGGATCCTTGCCCCGCCCCTTGAATTCCC-3′ (SEQ ID NO:25);

5′-GGGAATTCAAATGACGTCAAAAGGATCCAG-3′ (SEQ ID NO:26);

5′-CCTACCCACCCTGGATCCTTGCCCCGCCCCTTGAATTCCCAA

CCCTCCAC-3′ (SEQ ID NO:27);

5′-ATCCTTGCCCCGCCCCTTGAAT-3′ (SEQ ID NO:28); or

5′-TTGCCCCGCCCCTT (SEQ ID NO:29), wherein at least one of the cytosineresidues in SEQ ID NOs: 25-29 is substituted with 5-aza-cytosine.

For example, the oligonucleotide analogue may be

5′-CTGGATCCTTGCCCDGCCCCTTGAATTCCC-3′ (SEQ ID NO:30), wherein one of the14 cytosine residues in SEQ ID NO:25 at nucleotide position 15 issubstituted with 5-aza-cytosine.

In yet another aspect of the invention, an oligonucleotide analogue isprovided that is at least 6 nucleotide long, has at least one5-aza-cytosine as a base residue and adopts a hairpin conformation atambient temperature, such as 20-25° C., in aqueous solution, such aswater, saline, or a buffer comprising 20 mM HEPES (pH 7), 12% glycero, 1mM EDTA, 4 mM dithothreitol, 0.1% Nonidet P-40, and 3 mM MgCl₂.

In one embodiment, the oligonucleotide analogue has the followinggeneral secondary structure:

wherein N is any nucleotide; N′ is a nucleotide complementary to N; Z is5-aza-cytosine as a base residue; G is guanine as a base residue; 1, n,or m is an integer; nucleotide Nn, Nm, N′n, and N′m are positioned inthe stem region of the hairpin; and N₁ is positioned in the loop regionof the hairpin. Preferably, 1, n, or m is an integer greater than 2, 3,4, or 5. Optionally, 1 is 2, 3, 4, 5, or 6. Also optionally, if Nn, Nm,or N₁ has one or more cytosine residues, the cytosine residue issubstituted with 5-aza-cytosine.

In a particular embodiment, the oligonucleotide analogue (SEQ ID NO:31)has the following hairpin conformation:

wherein D is decitabine, A is adenosine or 2′-deoxyadenosine, T isthymidine or 2′-deoxythymidine, and C at nucleotide position 21 isoptionally substituted with 5-methyl-2′-deoxycytidine.

In any of above embodiments, the oligonucleotide analogue can besingle-stranded or double-stranded. When the oligonucleotide analogue isdouble-stranded, the first strand is the oligonucleotide analogue, andthe second strand may be an oligonucleotide with sequence complementaryto that of the first strand without the cytosine residue being replacedwith 5-aza-cytosine. For example, the first strand may be 5′-TTDGDGAA-3′(SEQ ID NO: 32) wherein D is decitabine; whereas the second strand maybe 5′-TTCGCGAA-3′ (SEQ ID NO: 33).

Optionally, when the second strand of oligonucleotide comprises one ormore cytosine residues, and at least one of the cytosine residues issubstituted with 5-methyl-cytosine.

Also optionally, when the first strand has a segment of 5′-Z-L-G-3′, andthe second strand comprises a segment of 3′-G-L-C′-5′ that matches withthe segment of 5′-Z-L-G-3′ in the first strand, wherein Z is5-aza-cytosine; G is guanine; L is a chemical linker covalently linkingZ and G, or G and C′; and C′ is 5-methyl-cytosine.

Also optionally, when the first strand has a segment of 5′-G-L-Z-3′, andthe second strand comprises a segment of 3′-C′-L-G-5′ that matches withthe segment of 5′-G-L-Z-3′ in the first strand, wherein Z is5-aza-cytosine; G is guanine; L is a chemical linker covalently linkingZ and G, or G and C′; and C′ is 5-methyl-cytosine.

The present invention also provides methods for synthesizing theoligonucleotide analogues and for modulating nucleic acid methylation.Also provided are phosphoramidite building blocks for synthesizing theoligonucleotide analogues, formulating and administering these compoundsor compositions to treat conditions, such as cancer and hematologicaldisorders.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows structure of decitabine, D (1a), cytosine, C (1a′), and5-methyl cytosine, mC (1a″).

FIG. 2A depicts examples of decitabine phosphoramidite building blocks.

FIG. 2B depicts decitabine phosphoramidite building block 1d, whereR₁=phenoxyacetyl.

FIG. 3A depicts 3′- and 5′-O-capped and controlled-pore glass 3′-linkeddecitabine derivatives.

FIG. 3B depicts protected decitabine 3′-linked onto controlled-poreglass support

FIG. 4 depicts examples of 3′-O-capped decitabine derivatives.

FIG. 5 illustrates a standard cycle for oligonucleotide synthesis.

FIG. 6A shows synthesis schemes of GpD dinucleotides andtetranucleotides, where X⁺ is a counter ion.

FIG. 6B depicts a model synthesis cycle of GpD dinucleotide (2a) andDpGpGpD tetranucleotide (3b).

FIG. 7 depicts DpGpGpD tetranucleotide.

FIG. 8 shows synthesis schemes of GpD dinucleotides andtetranucleotides.

FIG. 9 depicts GpDpG trinucleotide and GpDpDpG tetranucleotide.

FIG. 10 depicts protected decitabine 3′-linked onto poly (ethyleneglycol).

FIG. 11 shows synthesis schemes of 3′- and 5′-O-capped GpD dinucleotide.

FIG. 12 shows synthesis schemes of 3′- and 5′-O-capped DpG dinucleotide.

FIG. 13 depicts GpD and DpG dinucleotides with nuclease resistantphosphothioate linkage.

FIG. 14 depicts GpDpGpD and DpGpGpD tetranucleotides with nucleaseresistant phosphothioate linkage.

FIG. 15 depicts DpGpDpG and GpDpDpG tetranucleotides with nucleaseresistant phosphothioate linkage.

FIG. 16 depicts GpD and DpG dinucleotides with cytidine deaminaseresistant 4-amino groups.

FIG. 17 depicts GpD and DpG dinucleotides with cytidine deaminaseresistant 4-amino groups and nuclease resistant phosphothioate linkage.

FIG. 18 depicts GpDpGpD and DpGpGpD tetranucleotides with cytidinedeaminase resistant 4-amino groups.

FIG. 19 depicts GpDpGpD and DpGpGpD tetranucleotides with cytidinedeaminase resistant 4-amino groups and nuclease resistant phosphothioatelinkage.

FIG. 20 depicts Cap-O-GpD-O-Cap and Cap-O-DpG-O-Cap dinucleotides withcytidine deaminase resistant 4-amino groups.

FIG. 21 depicts Cap-O-GpD-O-Cap and Cap-O-DpG-O-Cap dinucleotides withcytidine deaminase resistant 4-amino groups and nuclease resistantphosphothioate linkage.

FIG. 22 depicts DpGpDpG and GpDpDpG tetranucleotides with cytidinedeaminase resistant 4-amino groups.

FIG. 23 depicts DpGpDpG and GpDpDpG tetranucleotides with cytidinedeaminase resistant 4-amino groups and nuclease resistant phosphothioatelinkage

FIG. 24A depicts -DpG-islets with natural phosphodiester backbone ormodified backbones.

FIG. 24B depicts a -DpG-islet peptide backbone.

FIG. 25 schematically illustrates a cell-based GFP assay for DNAmethylation Panel a) shows control cells; and panel b) cells treatedwith oligonucleotides of the present invention and expressing GFP.

FIG. 26 lists examples of inventive oligonucleotide analoguesspecifically targeting the promoter region of P15, BRAC1 or P16, where Dcan be decitabine or decitabine analogues and px=p for natural phosphatelinkage, px=ps for phosphorothioate linkage, px=bp for boranophospate,px=mp for methylphosphonate linkage.

FIG. 27 lists the sequence of P15 promoter region and examples ofsegments thereof, based on which DpG and GpD rich oligonucleotideanalogues can be made.

FIG. 28 lists the sequence of P16 promoter region and examples ofsegments thereof, based on which DpG and GpD rich oligonucleotideanalogues can be made.

FIG. 29 lists the sequence of BRCA1 promoter region and examples ofsegments thereof, based on which DpG and GpD rich oligonucleotideanalogues can be made.

FIG. 30 is a mass spectrum of GpD (2a) triethylammonium salt.

FIG. 31 is a mass spectrum of DpG (2b) triethylammonium salt.

FIG. 32 is a mass spectrum of DpGpGpD (3b) triethylammonium salt.

FIG. 33 is a mass spectrum of GpDpG (3c′) triethylammonium salt.

FIG. 34 is a mass spectrum of DpGpDpG (3c) triethylammonium salt.

FIG. 35 is a mass spectrum of phosphorothioate linked DpG (2f)triethylammonium salt.

FIG. 36 is a mass spectrum of DpG (2b) sodium salt.

FIG. 37 is a mass spectrum of HEG-DpG (2d) triethylammonium salt.

FIG. 38 is a mass spectrum of decitabine phosphooramidite building block(1d; R₁=phenoxyacetyl).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides oligonucleotide analogues whichincorporate an analogue of cytosine, 5-aza-cytosine, in theoligonucleotide sequence, e.g., in the form of 5-aza-2′-deoxycytidine(also known as decitabine) or 5-aza-cytidine (5-azaC). It is believedthat incorporation of one or more residues of 5-aza-cytosine into anoligonucleotide would have a DNA hypomethylation effect as replacementof cytosine with decitabine at a specific site of methylation producesan irreversible inactivation of DNA methyltransferase. Preferablydecitabine is incorporated into the oligonucleotide 5′-adjacent to aguanine residue to form a DpG islet in order to more specifically targetthe CpG islets of the human genome.

The invention is aimed to overcome potential toxicities associated withconventional hypomethylating agents such as decitabine and5-aza-cytidine. Compared to the free nucleoside forms, which arerandomly and extensively incorporated into the whole genome, theinventive compounds could act as primers and are incorporated mainlyinto the CpG-rich islands of the DNA during replication. Preferably, theinventive compounds act as primers and are incorporated specificallyinto the CpG-rich islands of the promoters of therapeutically ordiagnostically important genes, such as the tumor suppressor genes. Theinventive compounds could form temporarily hemimethylated stands withthe parental strand and function as the active trap of DNAmethyltransferases without being incorporated. The inventive compoundsmay also directly occupy and trap DNA methyltransferases without beingincorporated into the genome. Since DNA modification is localized to theCpG-rich islands in the promoter regions of tumor suppressor genes, whenthe inventive compounds are incorporated, the active trap is optimallyplaced and overall stability of the greater genome remainsuncompromised.

By modulating DNA methylation, the inventive compounds can be used astherapeutics, diagnostics as well as research reagents, especially inthe areas of cancer and hematological disorders. Aberranttranscriptional silencing of a number of genes, such as tumor suppressorgenes, is directly related to pathogenesis of cancer and other diseases.Due to methylation of cancer-related genes, expression of these genes issuppressed or completely silenced. Meanwhile, expression of these genesis required for induction of growth arrest, differentiation, and/orapoptotic cell death of transformed cells. Inaction of these genes inthe transformed cells leads to uncontrolled proliferation of thesecells, which eventually results in cancer. Thus, by using the inventivecompounds to actively trap DNA methyltransferases directly and withoutbeing incorporated into the genome or incorporated into the CpG-richislands of these genes, transcription of the genes can be reactivatedthrough inhibition of methylation of the promoters, thereby resultingsuppression of cancer cell proliferation.

The compounds of the present invention can also be useful for researchand diagnostics, because some embodiments of the inventive compounds canhybridize to a 5′-untranslated region or promoter sequence of a gene,enabling sandwich and other assays to easily be constructed to exploitthis fact. Hybridization of the oligonucleotide analogues of theinvention with the promoter sequence can be detected by means known inthe art. Such means may include conjugation or non-covalently binding ofan enzyme to the oligonucleotide analogue, radiolabelling of theoligonucleotide analogue or any other suitable detection means. Kitsusing such detection means for modulating activity of the promoter ofthe gene in a sample may also be prepared.

The present invention also provides methods for synthesizing theseoligonucleotide analogues and for modulating C-5 cytosine methylation.In particular, phosphoramidite building blocks and oligonucleotidescontaining decitabine (5-aza-2′-deoxycytidine; D), DpG-rich(Decitabine-phosphodiester linkage-Guanosine) islets and derivative, areprovided. Also provided are methods for preparing, formulating andadministering these compounds or compositions as therapeutics to a hostin need thereof. The inventive compounds, methods of synthesis,formulation of pharmaceutical compositions, preparation of vessels andkits, and use of the compounds or compositions for treating diseases orconditions are described in detail below.

1. Oligonucleotide Analogues of the Present Invention

In general the oligonucleotide analogue of the present invention has oneor more residues of 5-aza-cytosine (hereinafter abbreviated as “Z’)incorporated into an oligonucleotide sequence.

In one aspect of the invention, an isolated or synthetic oligonucleotideanalogue having 12 or less bases in length is provided, which comprisesone or more 5-aza-cytosine residues in the sequence of theoligonucleotide analogue.

In an embodiment, the oligonucleotide analogue has a general formula:—Z-L-G-, or -G-L-Z—,wherein Z is 5-aza-cytosine; G is guanine; and L is a chemical linkercovalently linking Z and G. The oligonucleotide analogue optionally hasmore than 30%, 35%, or 40% guanine residues in the sequence of theoligonucleotide analogue.

In particular embodiments, the oligonucleotide analogue is selected fromthe group consisting of 5′-DpG-3′, 5′-GpD-3′, 5′-DpGpD-3′, 5′-GpGpD-3′,5′-GpDpG-3′, 5′-GpDpD-3′, 5′-DpDpG-3′, 5′-DpGpG-3′, 5′-GpDpD-3′,5′-DpGpA-3′, 5′-DpGpDpG-3′, 5′-DpGpGpD-3′, 5′-GpDpGpD-3′, 5′-GpDpDpG-3′,5′-DpGpDpGpA-3′, wherein D is decitabine; p is a phospholinker; A is2′-deoxyadenosine, and G is 2′-deoxyguanosine.

In another aspect of the invention, an isolated or syntheticoligonucleotide analogue is provided which comprises, 2 or more copiesof a dinucleotide analogue having the general formula:—Z-L-G-, or -G-L-Z—,wherein Z is 5-aza-cytosine; G is guanine; and L is a chemical linkercovalently linking Z and G.

Optionally, the oligonucleotide analogue comprises less than 10, 8, 6,or 4 copies of the dinucleotide analogue —Z-L-G-, or -G-L-Z—.

In particular embodiments, the oligonucleotide analogue comprises asegment selected from the group consisting of 5′-DpG-3′, 5′-GpD-3′,5′-DpGpD-3′, 5′-GpGpD-3′, 5′-GpDpG-3′, 5′-GpDpD-3′, 5′-DpDpG-3′,5′-DpGpG-3′, 5′-GpDpD-3′, 5′-DpGpA-3′, 5′-DpGpDpG-3′, 5′-DpGpGpD-3′,5′-GpDpGpD-3′, 5′-GpDpDpG-3′, 5′-DpGpDpGpA-3′, wherein D is decitabine;p is a phospholinker; A is 2′-deoxyadenosine, and G is2′-deoxyguanosine.

In yet another aspect of the invention, an isolated or syntheticoligonucleotide analogue having at least 6 bases in length is provided,which comprises one or more 5-aza-cytosine residues in the sequence ofthe oligonucleotide analogue and has at least 75% sequence homology witha segment of a gene, preferably the 5′-untranslated region of a gene,such as the promoter of the gene.

In an embodiment, the oligonucleotide analogue has a general formula:—Z-L-G-, or -G-L-Z—,wherein Z is 5-aza-cytosine; G is guanine; and L is a chemical linkercovalently linking Z and G. The oligonucleotide analogue optionally hasmore than 30%, 35%, or 40% guanine residues in the sequence of theoligonucleotide analogue.

In particular embodiments, the oligonucleotide analogue comprises asegment selected from the group consisting of 5′-DpG-3′, 5′-GpD-3′,5′-DpGpD-3′, 5′-GpGpD-3′, 5′-GpDpG-3′, 5′-GpDpD-3′, 5′-DpDpG-3′,5′-DpGpG-3′, 5′-GpDpD-3′, 5′-DpGpA-3′, 5′-DpGpDpG-3′, 5′-DpGpGpD-3′,5′-GpDpGpD-3′, 5′-GpDpDpG-3′, 5′-DpGpDpGpA-3′, wherein D is decitabine;p is a phospholinker; A is 2′-deoxyadenosine, and G is2′-deoxyguanosine.

The gene is preferably a mammalian gene, and more preferably a humangene, and most preferably a human tumor suppressor gene. Examples of thehuman gene include, but are not limited to, VHL (the Von Hippon Landaugene involved in Renal Cell Carcinoma); P16/INK4A (involved inlymphoma); E-cadherin (involved in metastasis of breast, thyroid,gastric cancer); hMLH1 (involved in DNA repair in colon, gastric, andendometrial cancer); BRCA1 (involved in DNA repair in breast and ovariancancer); LKB1 (involved in colon and breast cancer); P15/INK4B (involvedin leukemia such as AML and ALL); ER (estrogen receptor, involved inbreast, colon cancer and leukemia); O6-MGMT (involved in DNA repair inbrain, colon, lung cancer and lymphoma); GST-pi (involved in breast,prostate, and renal cancer); TIMP-3 (tissue metalloprotease, involved incolon, renal, and brain cancer metastasis); DAPK1 (DAP kinase, involvedin apoptosis of B-cell lymphoma cells); P73 (involved in apoptosis oflymphomas cells); AR (androgen receptor, involved in prostate cancer);RAR-beta (retinoic acid receptor-beta, involved in prostate cancer);Endothelin-B receptor (involved in prostate cancer); Rb (involved incell cycle regulation of retinoblastoma); P14ARF (involved in cell cycleregulation); RASSF1 (involved in signal transduction); APC (involved insignal transduction); Caspase-8 (involved in apoptosis); TERT (involvedin senescence); TERC (involved in senescence); TMS-1 (involved inapoptosis); SOCS-1 (involved in growth factor response ofhepatocarcinoma); PITX2 (hepatocarcinoma breast cancer); MINT1; MINT2;GPR37; SDC4; MYOD1; MDR1; THBS1; PTC1; and pMDR1, as described inSantini et al. (2001) Ann. of Intern. Med. 134:573-586, which is hereinincorporated by reference in its entirety. Nucleotide sequences of thesegenes can be retrieved from the website of the National Center forBiotechnology Information (NCBI).

As examples, the promoter sequences of the tumor suppressor genes, p15,p16, and BRCA1, are shown in FIGS. 27, 28, and 29, respectively.Examples of oligonucleotide analogues with at least 75% sequencehomology with a segment of p15, p16, and BRCA1 are shown in FIGS. 27,28, and 29, respectively.

It is appreciated by skilled artisans in the field of nucleic acids thatthe higher degree of sequence homology of a tester polynucleotide withits target polynucleotide, the higher stringency of the condition whichthe tester polynucleotide can remain hybridized to the targetpolynucleotide. Thus, the oligonucleotide analogues of the presentinvention that are designed to target a specific gene are thoseoligonucleotide analogues that hybridize to the target gene under verylow to very high stringency conditions.

For oligonucleotide analogues of at least about 100 nucleotides inlength, very low to very high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and either 25% formamidefor very low and low stringencies, 35% formamide for medium andmedium-high stringencies, or 50% formamide for high and very highstringencies, following standard Southern blotting procedures. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS preferably at least at 45° C. (very low stringency),more preferably at least at 50° C. (low stringency), more preferably atleast at 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For shorter oligonucleotide analogues which are about 50 nucleotides toabout 100 nucleotides in length, stringency conditions are defined asprehybridization, hybridization, and washing post-hybridization at 5° C.to 10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures. The carrier material iswashed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15minutes using 6×SSC at 5° C. to 10° C. below the calculated T_(m).

Another non-limiting examples of high stringency conditions include ahybridization solution containing, e.g., about 5×SSC, 0.5% SDS, 100μg/ml denatured salmon sperm DNA and 50% formamide, at 42° C. Blots canbe washed at high stringency conditions that allow, e.g., for less than5% by mismatch (e.g., wash twice in 0.1% SSC and 0.1% SDS for 30 min at65° C.), i.e., selecting sequences having 95% or greater sequenceidentity.

Yet another non-limiting example of high stringency conditions includesa final wash at 65° C. in aqueous buffer containing 30 mM NaCl and 0.5%SDS. Another example of high stringent conditions is hybridization in 7%SDS, 0.5 M NaPO₄, pH 7, 1 mM EDTA at 50° C., e.g., overnight, followedby one or more washes with a 1% SDS solution at 42° C. Whereas highstringency washes can allow for less than 5% mismatch, reduced or lowstringency conditions can permit up to 20% nucleotide mismatch.Hybridization at low stringency can be accomplished as above, but usinglower formamide conditions, lower temperatures and/or lower saltconcentrations, as well as longer periods of incubation time.

In a preferred embodiment, the oligonucleotide analogue is capable ofhybridizing with the target gene under low stringency conditions. In amore preferred embodiment, the oligonucleotide analogue is capable ofhybridizing with the target gene under medium stringency conditions. Ina most preferred embodiment, the oligonucleotide analogue is capable ofhybridizing with the target gene under high stringency conditions.

In yet another aspect of the invention, an oligonucleotide analogue isprovided that binds an allosteric site on DNA methyltransferase therebyinhibiting DNA methyltransferase. The inhibition of DNAmethyltransferase prevents the methylation of DNA thereby treating thedisorder associated with aberrant DNA methylation, such as cancer andhematological disorders.

In one embodiment, the oligonucleotide analogue has a sequence of

5′-CTGGATCCTTGCCCCGCCCCTTGAATTCCC-3′ (SEQ ID NO:25);

5′-GGGAATTCAAATGACGTCAAAAGGATCCAG-3′ (SEQ ID NO:26);

5′-CCTACCCACCCTGGATCCTTGCCCCGCCCCTTGAATTCCCAA

CCCTCCAC-3′ (SEQ ID NO:27);

5′-ATCCTTGCCCCGCCCCTTGAAT-3′ (SEQ ID NO:28); or

5′-TTGCCCCGCCCCTT (SEQ ID NO:29), wherein at least one of the cytosineresidues in SEQ ID NOs: 25-28 is substituted with 5-aza-cytosine. Forexample, the oligonucleotide analogue may be

5′-CTGGATCCTTGCCCDGCCCCTTGAATTCCC-3′ (SEQ ID NO:30)

wherein one of the 14 cytosine residues in SEQ ID NO:25 at nucleotideposition 15 is substituted with 5-aza-cytosine. Other examples ofoligonucleotides that bind to DNA methyltransferase can be modifiedaccording to the present invention by substituting at least one of thecytosine residues can be found in WO 99/12027, which is hereinincorporated by reference in its entirety. The assays for testing theactivity of the oligonucleotide analogues of the present invention inbinding and inhibiting activity of DNA methyltransferase can also befound in WO 99/12027.

In yet another aspect of the invention, an oligonucleotide analogue isprovided that is at least 6 nucleotide long, has at least one5-aza-cytosine as a base residue and adopts a hairpin conformation atambient temperature, such as 20-25° C., in aqueous solution, such aswater, saline, or a buffer comprising 20 mM HEPES (pH 7), 12% glycero, 1mM EDTA, 4 mM dithothreitol, 0.1% Nonidet P-40, and 3 mM MgCl₂. It isbelieved that by adopting a hairpin conformation, the oligonucleotideanalogue better mimics the double-stranded DNA substrate for DNAmethyltransferase than a single-stranded oligonucleotide, thusinhibiting the activity of DNA methyltransferase more effectively.

In one embodiment, the oligonucleotide analogue has the followinggeneral secondary structure:

wherein N is any nucleotide; N′ is a nucleotide complementary to N; Z is5-aza-cytosine as a base residue; G is guanine as a base residue; l, n,or m is an integer; nucleotide Nn, Nm, N′n, and N′m are positioned inthe stem region of the hairpin; and N_(l) is positioned in the loopregion of the hairpin. Preferably, l, n, or m is an integer greater than2, 3, 4, or 5. Optionally, l is 2, 3, 4, 5, or 6. Also optionally, ifNn, Nm, or N_(l) has one or more cytosine residues, the cytosine residueis substituted with 5-aza-cytosine.

In a particular embodiment, the oligonucleotide analogue has thefollowing general secondary structure:

wherein C′ is 5-methyl-cytidine.

In another particular embodiment, the oligonucleotide analogue (SEQ IDNO:31) has the following hairpin conformation:

wherein D is decitabine A is adenosine or 2′-deoxyadenosine, T isthymidine or 2′-deoxythymidine, and C at nucleotide position 21 isoptionally substituted with 5-methyl-2′-deoxycytidine.

In any of above embodiments, the oligonucleotide analogue can besingle-stranded or double-stranded. When the oligonucleotide analogue isdouble-stranded, the first strand is the oligonucleotide analogue, andthe second strand may be an oligonucleotide with sequence complementaryto that of the first strand without the cytosine residue being replacedwith 5-aza-cytosine. For example, the first strand may be 5′-TTDGDGAA-3′(SEQ ID NO: 32) wherein D is decitabine; whereas the second strand maybe 5′-TTCGCGAA-3′ (SEQ ID NO: 33). Optionally, at least one of thecytosine residues in either the first or second strand may besubstituted with 5-methyl-cytosine.

In any of above embodiments, the linker between Z and G residues orbetween any two of the base residues in the oligonucleotide analogue ispreferably a sugar phosphorodiester linkage. Preferably the linker is aphosphorodiester linkage via 2′-deoxyribose or ribose, as in the naturalsugar phosphorodiester backbone in DNA and RNA, respectively.Optionally, to enhance the resistance to nuclease degradation in vivo,the natural phosphorodiester linker —O—P(═O)(O⁻)—O—CH₂— can be modifiedto be a phosphorothioate linker —O—P(═O)(S⁻)—O—CH₂—, bornophosphate ormethylphosphonate linker; the 2′-hydroxyl group of ribose can bemodified to be a 2′-methoxy group, 2′-methoxyethyl group, or 2′-fluorogroup. Examples of such oligonucleotide analogues with unnaturalbackbones are shown FIG. 24A where decitabine is linked to guanosinethrough ribose phosphate backbone. Also optionally, the natural sugarphosphorodiester backbone can be replaced with a protein nucleotide(PNA) backbone where the backbone is made from repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. An example ofsuch an oligonucleotide analogue with PNA backbone is shown in FIG. 24Bwhere 5-aza-cytosine is linked to guanine via a PNA backbone. Othertypes of linkers for oligonucleotides designed to be more resistant tonuclease degradation than the natural are described U.S. Pat. Nos.6,900,540 and 6,900,301, which are herein incorporated by reference.

The oligonucleotide analogues of the present invention may be onesisolated from biological sources, such as tissues, cells and body fluid,and preferably purified to a substantial degree of purity, morepreferably of at least 80% purity, and most preferably of at least 95%of purity. The oligonucleotide analogues may also be synthetic ones thatare non-naturally occurring oligonucleotide comprising a 5-aza-cytidine,e.g., chemically or enzymatically synthesized in vitro.

The oligonucleotide analogues of the present invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotide analogues of the invention may be prepared by formingone or more ester bond with any of the hydroxyl groups in the sugar ringusing an organic compound containing a carboxyl group, or as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention.

As used herein, a “pharmaceutical addition salt” includes apharmaceutically acceptable salt of an acid form of one of thecomponents of the compositions of the invention. These include organicor inorganic acid salts of the amines. Preferred acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotide analogues of the present invention, preferredexamples of pharmaceutically acceptable salts include but are notlimited to (a) salts formed with cations such as sodium, potassium,ammonium, magnesium, calcium, polyamines such as spermine andspermidine, etc.; (b) acid addition salts formed with inorganic acids,for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (c) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and (d) salts formed fromelemental anions such as chlorine, bromine, and iodine.

The invention also embraces isolated compounds. An isolated compoundrefers to a compound which represents at least 10%, preferably 20%, morepreferably 50% and most preferably 80% of the compound present in themixture, and exhibits a detectable (i.e. statistically significant)inhibitory activity of DNA methylation when tested in biological assayssuch as the combined bisulfite restriction analysis or COBRA (Xiong, Z.;Laird, P. W. Nucleic Acids Res. 1997, 25, 2532-2534) and radiolabeledmethyl incorporation assay (Francis, K. T.; Thompson, R. W.; Krumdieck,C. L. Am. J. Clin. Nutr. 1977, 30, 2028-2032).

2. Pharmaceutical Formulations of the Present Invention

According to the present invention, the oligonucleotide analogues orcompounds of the present invention can be formulated intopharmaceutically acceptable compositions for treating various diseasesand conditions.

The pharmaceutically-acceptable compositions of the present inventioncomprise one or more compounds of the invention in association with oneor more nontoxic, pharmaceutically-acceptable carriers and/or diluentsand/or adjuvants and/or excipients, collectively referred to herein as“carrier” materials, and if desired other active ingredients.

The compounds of the present invention are administered by any route,preferably in the form of a pharmaceutical composition adapted to such aroute, as illustrated below and are dependent on the condition beingtreated. The compounds and compositions can be, for example,administered orally, parenterally, intraperitoneally, intravenously,intraarterially, transdermally, sublingually, intramuscularly, rectally,transbuccally, intranasally, liposomally, via inhalation, vaginally,intraoccularly, via local delivery (for example by a catheter or stent),subcutaneously, intraadiposally, intraarticularly, or intrathecally.

The pharmaceutical formulation may optionally further include anexcipient added in an amount sufficient to enhance the stability of thecomposition, maintain the product in solution, or prevent side effects(e.g., potential ulceration, vascular irritation or extravasation)associated with the administration of the inventive formulation.Examples of excipients include, but are not limited to, mannitol,sorbitol, lactose, dextrox, cyclodextrin such as, α-, β-, andγ-cyclodextrin, and modified, amorphous cyclodextrin such ashydroxypropyl-, hydroxyethyl-, glucosyl-, maltosyl-, maltotriosyl-,carboxyamidomethyl-, carboxymethyl-, sulfobutylether-, anddiethylamino-substituted α, β-, and γ-cyclodextrin. Cyclodextrins suchas Encapsin® from Janssen Pharmaceuticals or equivalent may be used forthis purpose.

For oral administration, the pharmaceutical compositions can be in theform of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a therapeutically-effective amount of the activeingredient. Examples of such dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules which can contain, inaddition to the active ingredient, conventional carriers such as bindingagents, for example, acacia gum, gelatin, polyvinylpyrrolidone,sorbitol, or tragacanth; fillers, for example, calcium phosphate,glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, forexample, magnesium stearate, polyethylene glycol, silica, or talc;disintegrants, for example, potato starch, flavoring or coloring agents,or acceptable wetting agents. Oral liquid preparations generally are inthe form of aqueous or oily solutions, suspensions, emulsions, syrups orelixirs may contain conventional additives such as suspending agents,emulsifying agents, non-aqueous agents, preservatives, coloring agentsand flavoring agents. Examples of additives for liquid preparationsinclude acacia, almond oil, ethyl alcohol, fractionated coconut oil,gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin,methyl cellulose, methyl or propyl para-hydroxybenzoate, propyleneglycol, sorbitol, or sorbic acid.

For topical use the compounds of the present invention can also beprepared in suitable forms to be applied to the skin, or mucus membranesof the nose and throat, and can take the form of creams, ointments,liquid sprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient.

For application to the eyes or ears, the compounds of the presentinvention can be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention can beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride.

Alternatively, the compounds of the present invention can be in powderform for reconstitution in the appropriate pharmaceutically acceptablecarrier at the time of delivery.

The pharmaceutical compositions can be administered via injection.Formulations for parenteral administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions or suspensions can be prepared from sterile powders orgranules having one or more of the carriers mentioned for use in theformulations for oral administration. The compounds can be dissolved inpolyethylene glycol, propylene glycol, ethanol, corn oil, benzylalcohol, sodium chloride, and/or various buffers.

The oligonucleotide analogues of the present invention may be preparedand formulated as emulsions. Emulsions are typically heterogenoussystems of one liquid dispersed in another in the form of dropletsusually exceeding 0.1 μm in diameter. (Idson, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the oligonucleotideanalogues are formulated as microemulsions. A microemulsion may bedefined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Besides microemulsions there are many organized surfactant structuresthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G.sub.M1, or (B)is derivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G.sub.M1,galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C.sub.12 15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

In a particular embodiment, the compounds of the present invention canbe formulated into a pharmaceutically acceptable composition comprisingthe compound solvated in non-aqueous solvent that includes glycerin,propylene glycol, polyethylene glycol, or combinations thereof. It isbelieved that the compounds will be stable in such pharmaceuticalformulations so that the pharmaceutical formulations may be stored for aprolonged period of time prior to use.

In current clinical treatment with decitabine, to minimize drugdecomposition decitabine is supplied as lyophilized powder andreconstituted in a cold aqueous solution containing water in at least40% vol of the solvent, such as WFI, and diluted in cold infusion fluidsprior to administration. Such a formulation and treatment regimensuffers from a few drawbacks. First, refrigeration of decitabine in coldsolution becomes essential, which is burdensome in handling andeconomically less desirable than a formulation that can sustain storageat higher temperatures. Second, due to rapid decomposition of decitabinein aqueous solution, the reconstituted decitabine solution may only beinfused to a patient for a maximum of 3 hr if the solution has beenstored in the refrigerator for less than 7 hr. In addition, infusion ofcold fluid can cause great discomfort and pain to the patient, whichinduces the patient's resistance to such a regimen.

By modifying the triazine ring and/or the ribose ring of decitabine andby formulating the compound with non-aqueous solvent, the pharmaceuticalformulations can circumvent the above-listed problems associated withthe current clinical treatment with decitabine. These formulations ofthe inventive compounds are believed to be more chemically stable thandecitabine formulated in aqueous solutions containing water in at least40% vol. of the solvent.

In a preferred embodiment, the inventive formulation contains less than40% water in the solvent, optionally less than 20% water in the solvent,optionally less than 10% water in the solvent, or optionally less than1% water in the solvent. In one variation, the pharmaceuticalformulation is stored in a substantially anhydrous form. Optionally, adrying agent may be added to the pharmaceutical formulation to absorbwater.

Owing to the enhanced stability, the inventive formulation may be storedand transported at ambient temperature, thereby significantly reducingthe cost of handling the drug. Further, the inventive formulation may beconveniently stored for a long time before being administered to thepatient. In addition, the inventive formulation may be diluted withregular infusion fluid (without chilling) and administered to a patientat room temperature, thereby avoiding causing patients' discomfortassociated with infusion of cold fluid.

In another embodiment, the inventive compound is dissolved in glycerinat different concentrations. For example, the formulation may optionallycomprise between 0.1 and 200; between 1 and 100; between 1 and 50;between 2 and 50; between 2 and 100; between 5 and 100; between 10 and100 or between 20 and 100 mg inventive compound per ml of glycerin.Specific examples of the inventive compound per glycerin concentrationsinclude but are not limited to 2, 5, 10, 20, 22, 25, 30, 40 and 50mg/ml.

Different grades of glycerin (synonyms: 1,2,3-propanetriol; glycerol;glycol alcohol; glycerol anhydrous) may be used to prepare theformulations. Preferably, glycerin with chemical purity higher than 90%is used to prepare the formulations.

In another embodiment, the inventive compound is dissolved in propyleneglycol at different concentrations. For example, the formulation mayoptionally comprise between 0.1 and 200; between 0.1 and 100; between0.1 and 50; between 2 and 50; between 2 and 100; between 5 and 100;between 10 and 100 or between 20 and 100 mg inventive compound per ml ofpropylene glycol. Specific examples of decitabine per propylene glycolconcentrations include but are not limited to 2, 5, 10, 20, 22, 25, 30,40 and 50 mg/ml.

In yet another embodiment, the inventive compound is dissolved in asolvent combining glycerin and propylene glycol at differentconcentrations. The concentration of propylene glycol in the solvent isbetween 0.1-99.9%, optionally between 1-90%, between 10-80%, or between50-70%.

In yet another embodiment, the inventive compound is dissolved atdifferent concentrations in a solvent combining glycerin andpolyethylene glycol (PEG) such as PEG300, PEG400 and PEG1000. Theconcentration of polyethylene glycol in the solvent is between0.1-99.9%, optionally between 1-90%, between 10-80%, or between 50-70%.

In yet another embodiment, the inventive compound is dissolved atdifferent concentrations in a solvent combining propylene glycol,polyethylene glycol and glycerin. The concentration of propylene glycolin the solvent is between 0.1-99.9%, optionally between 1-90%, between10-60%, or between 20-40%; and the concentration of polyethylene glycolin the solvent is between 0.1-99.9%, optionally between 1-90%, between10-80%, or between 50-70%.

It is believed and experimentally proven that addition of propyleneglycol can further improve chemical stability, reduce viscosity of theformulations and facilitate dissolution of the inventive compound in thesolvent.

The pharmaceutical formulation may further comprise an acidifying agentadded to the formulation in a proportion such that the formulation has aresulting pH between about 4 and 8. The acidifying agent may be anorganic acid. Examples of organic acid include, but are not limited to,ascorbic acid, citric acid, tartaric acid, lactic acid, oxalic acid,formic acid, benzene sulphonic acid, benzoic acid, maleic acid, glutamicacid, succinic acid, aspartic acid, diatrizoic acid, and acetic acid.The acidifying agent may also be an inorganic acid, such as hydrochloricacid, sulphuric acid, phosphoric acid, and nitric acid.

It is believed that adding an acidifying agent to the formulation tomaintain a relatively neutral pH (e.g., within pH 4-8) facilitates readydissolution of the inventive compound in the solvent and enhanceslong-term stability of the formulation. In alkaline solution, there is arapid reversible decomposition of decitabine toN-(formylamidino)-N′-β-D-2-deoxyribofuranosylurea, which decomposesirreversibly to form 1-β-D-2′-deoxyribofuranosyl-3-guanylurea. The firststage of the hydrolytic degradation involves the formation ofN-amidinium-N′-(2-deoxy-β-D-erythropentofuranosyl)urea formate (AUF).The second phase of the degradation at an elevated temperature involvesformation of guanidine. In acidic solution,N-(formylamidino)-N′-β-D-2-deoxyribofuranosylurea and some unidentifiedcompounds are formed. In strongly acidic solution (at pH <2.2)5-azacytosine is produced. Thus, maintaining a relative neutral pH maybe advantageous for the formulation comprising the analogs andderivatives of decitabine.

In a variation, the acidifying agent is ascorbic acid at a concentrationof 0.01-0.2 mg/ml of the solvent, optionally 0.04-0.1 mg/ml or 0.03-0.07mg/ml of the solvent.

The pH of the pharmaceutical formulation may be adjusted to be betweenpH 4 and pH 8, preferably between pH 5 and pH 7, and more preferablybetween pH 5.5 and pH 6.8.

The pharmaceutical formulation is preferably at least 80%, 90%, 95% ormore stable upon storage at 25° C. for 7, 14, 21, 28 or more days. Thepharmaceutical formulation is also preferably at least 80%, 90%, 95% ormore stable upon storage at 40° C. for 7, 14, 21, 28 or more days.

In one embodiment, the pharmaceutical formulation of the presentinvention is prepared by taking glycerin and dissolving the inventivecompound in the glycerin. This may be done, for example, by adding theinventive compound to the glycerin or by adding the glycerin todecitabine. By their admixture, the pharmaceutical formulation isformed.

Optionally, the method further comprises additional steps to increasethe rate at which the inventive compound is solvated by the glycerin.Examples of additional steps that may be performed include, but are norlimited to, agitation, heating, extension of solvation period, andapplication of micronized inventive compound and the combinationsthereof.

In one variation, agitation is applied. Examples of agitation includebut are nor limited to, mechanical agitation, sonication, conventionalmixing, conventional stirring and the combinations thereof. For example,mechanical agitation of the formulations may be performed according tomanufacturer's protocols by Silverson homogenizer manufactured bySilverson Machines Inc., (East Longmeadow, Mass.).

In another variation, heat may be applied. Optionally, the formulationsmay be heated in a water bath. Preferably, the temperature of the heatedformulations may be less than 70° C., more preferably, between 25° C.and 40° C. As an example, the formulation may be heated to 37° C.

In yet another variation, the inventive compound is solvated in glycerinover an extended period of time.

In yet another variation, a micronized form of the inventive compoundmay also be employed to enhance solvation kinetics. Optionally,micronization may be performed by a milling process. As an example,micronization may be performed by milling process performedMastersizerusing an Air Jet Mill, manufactured by IncFluid Energy AljetInc. (Boise, Id. Telford, Pa.).

Optionally, the method further comprises adjusting the pH of thepharmaceutical formulations by commonly used methods. In one variation,pH is adjusted by addition of acid, such as ascorbic acid, or base, suchas sodium hydroxide. In another variation, pH is adjusted and stabilizedby addition of buffered solutions, such as solution of(Ethylenedinitrilo) tetraacetic acid disodium salt (EDTA). As decitabineis known to be pH-sensitive, adjusting the pH of the pharmaceuticalformulations to approximately pH 7 may increase the stability oftherapeutic component.

Optionally, the method further comprises separation of non-dissolvedinventive compound from the pharmaceutical formulations. Separation maybe performed by any suitable technique. For example, a suitableseparation method may include one or more of filtration, sedimentation,and centrifugation of the pharmaceutical formulations. Clogging that maybe caused by non-dissolved particles of the inventive compound, maybecome an obstacle for administration of the pharmaceutical formulationsand a potential hazard for the patient. The separation of non-dissolvedinventive compound from the pharmaceutical formulations may facilitateadministration and enhance safety of the therapeutic product.

Optionally, the method further comprises sterilization of thepharmaceutical formulations. Sterilization may be performed by anysuitable technique. For example, a suitable sterilization method mayinclude one or more of sterile filtration, chemical, irradiation, heat,and addition of a chemical disinfectant to the pharmaceuticalformulation.

As noted, decitabine is unstable in water and hence it may be desirableto reduce the water content of the glycerin used for formulating theinventive compound. Accordingly, prior to the dissolution and/orsterilization step, the glycerin may be dried. Such drying of glycerinor the solution of the inventive compound in glycerin may be achieved bythe addition of a pharmaceutically acceptable drying agent to theglycerin. The glycerin or the inventive formulations may be dried, forexample by filtering it through a layer comprising a drying agent.

Optionally, the method may further comprise adding one or more membersof the group selected from drying agents, buffering agents,antioxidants, stabilizers, antimicrobials, and pharmaceutically inactiveagents. In one variation, antioxidants such as ascorbic acid, ascorbatesalts and mixtures thereof may be added. In another variationstabilizers like glycols may be added.

3. Vessels or Kits Containing Inventive Compounds or Formulations

The pharmaceutical formulations, described in this invention, may becontained in a sterilized vessel such as syringes, vials or ampoules ofvarious sizes and capacities. The sterilized vessel may optionallycontain between 1-50 ml, 1-25 ml or 1-20 ml or 1-10 ml of theformulations. Sterilized vessels maintain sterility of thepharmaceutical formulations, facilitate transportation and storage, andallow administration of the pharmaceutical formulations without priorsterilization step.

The present invention also provides a kit for administering theinventive compound to a host in need thereof. In one embodiment, the kitcomprises the inventive compound in a solid, preferably powder form, anda non-aqueous diluent that comprises glyercin, propylene glycol,polyethylene glycol, or combinations thereof. Mixing of the soliddecitabine and the diluent preferably results in the formation of apharmaceutical formulation according to the present invention. Forexample, the kit may comprise a first vessel comprising the inventivecompound in a solid form; and a vessel container comprising a diluentthat comprises glyercin; wherein adding the diluent to the solidinventive compound results in the formation of a pharmaceuticalformulation for administering the inventive compound. Mixing the solidthe inventive compound and diluent may optionally form a pharmaceuticalformulation that comprises between 0.1 and 200 mg of the inventivecompound per ml of the diluent, optionally between 0.1 and 100, between2 mg and 50 mg, 5 mg and 30 mg, between 10 mg and 25 mg per ml of thesolvent.

According to the embodiment, the diluent is a combination of propyleneglycol and glycerin, wherein the concentration of propylene glycol inthe solvent is between 0.1-99.9%, optionally between 1-90%, between10-60%, or between 20-40%.

Also according to the embodiment, the diluent is a combination ofpolyethylene glycol and glycerin, wherein the concentration ofpolyethylene glycol in the solvent is between 0.1-99.9%, optionallybetween 1-90%, between 10-60%, or between 20-40%.

Also according to the embodiment, the diluent is a combination ofpropylene glycol, polyethylene glycol and glycerin, wherein theconcentration of propylene glycol in the solvent is between 0.1-99.9%,optionally between 1-90%, between 10-60%, or between 20-40%; and theconcentration of polyethylene glycol in the solvent is between0.1-99.9%, optionally between 1-90%, between 10-60%, or between 20-40%.

The diluent also optionally comprises 40%, 20%, 10%, 5%, 2% or lesswater. In one variation, the diluent is anhydrous and may optionallyfurther comprise a drying agent. The diluent may also optionallycomprise one or more drying agents, glycols, antioxidants and/orantimicrobials.

The kit may optionally further include instructions. The instructionsmay describe how the solid the inventive compound and the diluent shouldbe mixed to form a pharmaceutical formulation. The instructions may alsodescribe how to administer the resulting pharmaceutical formulation to apatient. It is noted that the instructions may optionally describe theadministration methods according to the present invention.

The diluent and the inventive compound may be contained in separatevessels. The vessels may come in different sizes. For example, thevessel may comprise between 1 and 50, 1 and 25, 1 and 20, or 1 and 10 mlof the diluent.

The pharmaceutical formulations provided in vessels or kits may be in aform that is suitable for direct administration or may be in aconcentrated form that requires dilution relative to what isadministered to the patient. For example, pharmaceutical formulations,described in this invention, may be in a form that is suitable fordirect administration via infusion.

The methods and kits described herein provide flexibility whereinstability and therapeutic effect of the pharmaceutical formulationscomprising the inventive compound may be further enhanced orcomplemented.

4. Methods for Administrating Inventive Compounds/Compositions

The compounds/formulations of the present invention can be administeredby any route, preferably in the form of a pharmaceutical compositionadapted to such a route, as illustrated below and are dependent on thecondition being treated. The compounds or formulations can be, forexample, administered orally, parenterally, topically,intraperitoneally, intravenously, intraarterially, transdermally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery (for example by catheter or stent), subcutaneously,intraadiposally, intraarticularly, or intrathecally. The compoundsand/or compositions according to the invention may also be administeredor co-administered in slow release dosage forms.

The compounds and/or compositions of this invention may be administeredor co-administered in any conventional dosage form. Co-administration inthe context of this invention is defined to mean the administration ofmore than one therapeutic agent in the course of a coordinated treatmentto achieve an improved clinical outcome. Such co-administration may alsobe coextensive, that is, occurring during overlapping periods of time.

The inventive compound or the composition containing the inventivecompound may be administered into a host such as a patient at a dose of0.1-1000 mg/m², optionally 1-200 mg/m², optionally 1-50 mg/m²,optionally 1-40 mg/m², optionally 1-30 mg/m², optionally 1-20 mg/m², oroptionally 5-30 mg/m².

For example, the compound/composition of the present invention may besupplied as sterile powder for injection, together with buffering saltsuch as potassium dihydrogen and pH modifier such as sodium hydroxide.This formulation is preferably stored at 2 8° C., which should keep thedrug stable for at least 2 years. This powder formulation may bereconstituted with 10 ml of sterile water for injection. This solutionmay be further diluted with infusion fluid known in the art, such as0.9% sodium chloride injection, 5% dextrose injection and lactatedringer's injection. It is preferred that the reconstituted and dilutedsolutions be used within 4-6 hours for delivery of maximum potency.

In a preferred embodiment, the inventive compound/composition isadministered to a patient by injection, such as subcutaneous injection,bolus i.v. injection, continuous i.v. infusion and i.v. infusion over 1hour. Optionally the inventive compound/composition is administered to apatient via an 1-24 hour i.v. infusion per day for 3-5 days pertreatment cycle at a dose of 0.1-1000 mg/m² per day, optionally at adose of 1-100 mg/m² per day, optionally at a dose of 2-50 mg/m² per day,optionally at a dose of 10-30 mg/m² per day, or optionally at a dose of5-20 mg/m² per day,

For decitabine or azacitidine, the dosage below 50 mg/m² is consideredto be much lower than that used in conventional chemotherapy for cancer.By using such a low dose of the analog/derivative of decitabine orazacitidine, transcriptional activity of genes silenced in the cancercells by aberrant methylation can be activated to trigger downstreamsignal transduction, leading to cell growth arrest, differentiation andapoptosis, which eventually results in death of these cancer cells. Thislow dosage, however, should have less systemic cytotoxic effect onnormal cells, and thus have fewer side effects on the patient beingtreated.

The pharmaceutical formulations may be co-administered in anyconventional form with one or more member selected from the groupcomprising infusion fluids, therapeutic compounds, nutritious fluids,anti-microbial fluids, buffering and stabilizing agents.

As described above, the inventive compounds can be formulated in aliquid form by solvating the inventive compound in a non-aqueous solventsuch as glycerin. The pharmaceutical liquid formulations provide thefurther advantage of being directly administrable, (e.g., withoutfurther dilution) and thus can be stored in a stable form untiladministration. Further, because glycerin can be readily mixed withwater, the formulations can be easily and readily further diluted justprior to administration. For example, the pharmaceutical formulationscan be diluted with water 180, 60, 40, 30, 20, 10, 5, 2, 1 minute orless before administration to a patient.

Patients may receive the pharmaceutical formulations intravenously. Thepreferred route of administration is by intravenous infusion.Optionally, the pharmaceutical formulations of the current invention maybe infused directly, without prior reconstitution.

In one embodiment, the pharmaceutical formulation is infused through aconnector, such as a Y site connector, that has three arms, eachconnected to a tube. As an example, Baxter® Y-connectors of varioussizes can be used. A vessel containing the pharmaceutical formulation isattached to a tube further attached to one arm of the connector.Infusion fluids, such as 0.9% sodium chloride, or 5% dextrose, or 5%glucose, or Lactated Ringer's, are infused through a tube attached tothe other arm of the Y-site connector. The infusion fluids and thepharmaceutical formulations are mixed inside the Y site connector. Theresulting mixture is infused into the patient through a tube connectedto the third arm of the Y site connector. The advantage of thisadministration approach over the prior art is that the inventivecompound is mixed with infusion fluids before it enters the patient'sbody, thus reducing the time when decomposition of the inventivecompound may occur due to contact with water. For example, the inventivecompound is mixed less than 10, 5, 2 or 1 minutes before entering thepatient's body.

Patients may be infused with the pharmaceutical formulations for 1, 2,3, 4, 5 or more hours, as a result of the enhanced stability of theformulations. Prolonged periods of infusion enable flexible schedules ofadministration of therapeutic formulations.

Alternatively or in addition, speed and volume of the infusion can beregulated according to the patient's needs. The regulation of theinfusion of the pharmaceutical formulations can be performed accordingto existing protocols.

The pharmaceutical formulations may be co-infused in any conventionalform with one or more member selected from the group comprising infusionfluids, therapeutic compounds, nutritious fluids, anti-microbial fluids,buffering and stabilizing agents. Optionally, therapeutic componentsincluding, but are not limited to, anti-neoplastic agents, alkylatingagents, agents that are members of the retinoids superfamily, antibioticagents, hormonal agents, plant-derived agents, biologic agents,interleukins, interferons, cytokines, immuno-modulating agents, andmonoclonal antibodies, may be co-infused with the inventiveformulations.

Co-infusion in the context of this invention is defined to mean theinfusion of more than one therapeutic agents in a course of coordinatedtreatment to achieve an improved clinical outcome. Such co-infusion maybe simultaneous, overlapping, or sequential. In one particular example,co-infusion of the pharmaceutical formulations and infusion fluids maybe performed through Y-type connector.

The pharmacokinetics and metabolism of intravenously administered thepharmaceutical formulations resemble the pharmacokinetics and metabolismof intravenously administered the inventive compound.

In humans, decitabine displayed a distribution phase with a half-life of7 minutes and a terminal half-life on the order of 10-35 minutes asmeasured by bioassay. The volume of distribution is about 4.6 L/kg. Theshort plasma half-life is due to rapid inactivation of decitabine bydeamination by liver cytidine deaminase. Clearance in humans is high, onthe order of 126 mL/min/kg. The mean area under the plasma curve in atotal of 5 patients was 408 μg/h/L with a peak plasma concentration of2.01 μM. In patients decitabine concentrations were about 0.4 μg/ml (2μM) when administered at 100 mg/m² as a 3-hour infusion. During a longerinfusion time (up to 40 hours) plasma concentration was about 0.1 to 0.4μg/mL. With infusion times of 40-60 hours, at an infusion rate of 1mg/kg/h, plasma concentrations of 0.43-0.76 μg/mL were achieved. Thesteady-state plasma concentration at an infusion rate of 1 mg/kg/h isestimated to be 0.2-0.5 μg/mL. The half-life after discontinuing theinfusion is 12-20 min. The steady-state plasma concentration ofdecitabine was estimated to be 0.31-0.39 μg/mL during a 6-hour infusionof 100 mg/m². The range of concentrations during a 600 mg/m² infusionwas 0.41-16 μg/mL. Penetration of decitabine into the cerebrospinalfluid in man reaches 14-21% of the plasma concentration at the end of a36-hour intravenous infusion. Urinary excretion of unchanged decitabineis low, ranging from less than 0.01% to 0.9% of the total dose, andthere is no relationship between excretion and dose or plasma druglevels. High clearance values and a total urinary excretion of less than1% of the administered dose suggest that decitabine is eliminatedrapidly and largely by metabolic processes.

Owing to their enhanced stability in comparison with decitabine, theinventive compounds/compositions can enjoy longer shelf life when storedand circumvent problems associated with clinical use of decitabine. Forexample, the inventive compounds may be supplied as lyophilized powder,optionally with an excipient (e.g., cyclodextrin), acid (e.g., ascorbicacid), alkaline (sodium hydroxide), or buffer salt (monobasic potassiumdihydrogen phosphate). The lyophilized powder can be reconstituted withsterile water for injection, e.g., i.v., i.p., i.m., or subcutaneously.Optionally, the powder can be reconstituted with aqueous or non-aqueoussolvent comprising a water miscible solvent such as glycerin, propyleneglycol, ethanol and PEG. The resulting solution may be administereddirectly to the patient, or diluted further with infusion fluid, such as0.9% Sodium Chloride; 5% Dextrose; 5% Glucose; and Lactated Ringer'sinfusion fluid.

The inventive compounds/compositions may be stored under ambientconditions or in a controlled environment, such as under refrigeration(2-8° C.; 36-46° F.). Due to their superior stability in comparison withdecitabine, the inventive compounds/compositions can be stored at roomtemperature, reconstituted with injection fluid, and administered to thepatient without prior cooling of the drug solution.

In addition, due to their enhanced chemical stability, the inventivecompound/composition should have a longer plasma half-life compared tothat of decitabine. Thus, the inventive compound/composition may beadministered to the patient at a lower dose and/or less frequently thanthat for decitabine.

5. Combination Therapy with Inventive Pharmaceutical Compositions

The compounds or pharmaceutical formulations of the present inventionmay be used in conjunction with inhibitors of histone deacetylase (HDAC)to further modulate transcription of genes, e.g., to reestablishtranscription of genes silenced by hypermethylation and acetylation ofhistones, in a synergistic manner.

HDAC plays important roles in transcription silencing of genes. Theamount of acetylation on the histones is controlled by the opposingactivities of two types of enzymes, histone acetyl transferase (HATs)and histone deacetylases (HDACs). Substrates for these enzymes includee-amino groups of lysine residues located in the amino-terminal tails ofthe histones H3, H4, H2A, and H2B. These amino acid residues areacetylated by HATs and deacetylated by HDACs. With the removal of theacetyl groups from the histone lysine by HDACs, a positive charge isrestored to the lysine residue, thereby condensing the structure ofnucleosome and silencing the genes contained within. Thus, to activatethese genes silenced by deacetylase of histones, the activity of HADCsshould be inhibited. With the inhibition of HDAC, histones areacetylated and the DNA that is tightly wrapped around a deacetylatedhistone core relaxes. The opening of DNA conformation leads toexpression of specific genes.

In addition to deacelation of histones, HDACs may also regulated geneexpression by deacetylating transcription factors, such as p53 (a tumorsuppressor gene), GATA-1, TFIIE, and TFIIF. Gu and Roeder (1997) Cell90:595-606 (p53); and Boyes et al. (1998) Nature 396:594-598 (GATA-1).HDACs also participate in cell cycle regulation, for example, bytranscription repression which is mediated by RB tumor suppressorproteins recruiting HDACs. Brehm et al. (1998) Nature 391:597-601. Thus,inhibition of HDACs should activate expression of tumor suppressor genessuch as p53 and RB and as a result promote cell growth arrest,differentiation and apoptosis induced by these genes.

As described above, aberrant transcriptional silencing of a number ofgenes, such as tumor suppressor genes, is directly related topathogenesis of cancer and other diseases. Methylation of cytosineresidues in DNA and removal of acetyl groups from histones are the twoprimary mechanisms for gene silencing. Due to methylation and/or histonedeacetylase of cancer-related genes, expression of these genes issuppressed or completely silenced. Meanwhile, expression of these genesis required for induction of growth arrest, differentiation, and/orapoptotic cell death of transformed cells. Inaction of these genes inthe transformed cells leads to uncontrolled proliferation of thesecells, which eventually results in cancer.

By combining the inventive compounds/compositions with HDAC inhibitors,genes required for induction of growth arrest, differentiation and celldeath of transformed cells can be reactivated effectively. The inventivecompounds/compositions inhibit methylation of DNA for the genes,especially in the regulatory region, thus resulting in activation oftranscription of the gene. Meanwhile, HDAC inhibitors inhibitdeacetylase of the histones in the nucleosomal core of the gene, thusresulting in net increase of the acetylation of histones, which, inturn, activates transcription of the gene. By exploiting these twocomplementary mechanisms, the combination therapy may reestablish genetranscription more effectively and, ideally, in a synergistic manner. Acombination therapy having synergistic effects should require a lessamount of each inhibitor than it being used alone, thus reducingpotential side effects associated systemic administration of highdosages of the inhibitors and improving therapeutic index.

Many anticancer agents exert their anti-cancer effects by triggeringsignal transduction cascades involving proteins encoded by these tumorsuppressor genes. With insufficient expression of these genes in cancercells, the anti-cancer effects of these anti-neoplastic agents may beseverely reduced or completely eradicated. Through reactivation orre-expression of these genes that are epigenetically silenced by DNAmethylation and histone deacetylase, the intrinsic defense mechanisms ofthe body are mobilized to combat the disease by restoration of thetumor-suppressing functions to cancer cells in response to signals sentby the anti-cancer agent administered. Such stimulation of the intrinsictumor suppressing functions of the body should lead to the requirementof lower dosage of the anticancer agent, thus resulting in a highertherapeutic index (i.e., greater efficacy and lower toxicity) of theagent.

Inhibitors of HDACs include, but are not limited to, the followingstructural classes: 1) hydroxamic acids, 2) cyclic peptides, 3)benzamides, and 4) short-chain fatty acids.

Examples of hydroxamic acids and hydroxamic acid derivatives, but arenot limited to, trichostatin A (TSA), suberoylanilide hydroxamic acid(SAHA), oxamflatin, suberic bishydroxamic acid (SBHA),m-carboxy-cinnamic acid bishydroxamic acid (CBHA), and pyroxamide. TSAwas isolated as an antifungi antibiotic (Tsuji et al (1976) J. Antibiot(Tokyo) 29:1-6) and found to be a potent inhibitor of mammalian HDAC(Yoshida et al. (1990) J. Biol. Chem. 265:17174-17179). The finding thatTSA-resistant cell lines have an altered HDAC evidences that this enzymeis an important target for TSA. Other hydroxamic acid-based HDACinhibitors, SAHA, SBHA, and CBHA are synthetic compounds that are ableto inhibit HDAC at micromolar concentration or lower in vitro or invivo. Glick et al. (1999) Cancer Res. 59:4392-4399. These hydroxamicacid-based HDAC inhibitors all possess an essential structural feature:a polar hydroxamic terminal linked through a hydrophobic methylenespacer (e.g. 6 carbon at length) to another polar site which is attachedto a terminal hydrophobic moiety (e.g., benzene ring). Compoundsdeveloped having such essential features also fall within the scope ofthe hydroxamic acids that may be used as HDAC inhibitors.

Cyclic peptides used as HDAC inhibitors are mainly cyclic tetrapeptides.Examples of cyclic peptides include, but are not limited to, trapoxin A,apicidin and FR901228. Trapoxin A is a cyclic tetrapeptide that containsa 2-amino-8-oxo-9,10-epoxy-decanoyl (AOE) moiety. Kijima et al. (1993)J. Biol. Chem. 268:22429-22435. Apicidin is a fungal metabolite thatexhibits potent, broad-spectrum antiprotozoal activity and inhibits HDACactivity at nanomolar concentrations. Darkin-Rattray et al. (1996) Proc.Natl. Acad. Sci. USA. 93; 13143-13147. FR901228 is a depsipeptide thatis isolated from Chromobacterium violaceum, and has been shown toinhibit HDAC activity at micromolar concentrations.

Examples of benzamides include but are not limited to MS-27-275. Saitoet al. (1990) Proc. Natl. Acad. Sci. USA. 96:4592-4597. Examples ofshort-chain fatty acids include but are not limited to butyrates (e.g.,butyric acid, arginine butyrate and phenylbutyrate (PB)). Newmark et al.(1994) Cancer Lett. 78:1-5; and Carducci et al. (1997) Anticancer Res.17:3972-3973. In addition, depudecin which has been shown to inhibitHDAC at micromolar concentrations (Kwon et al. (1998) Proc. Natl. Acad.Sci. USA. 95:3356-3361) also falls within the scope of histonedeacetylase inhibitor of the present invention.

The compounds or pharmaceutical formulations of the present inventionmay also be used in conjunction with other therapeutic componentsincluding but not limiting to anti-neoplastic agents, alkylating agents,agents that are members of the retinoids superfamily, antibiotic agents,hormonal agents, plant-derived agents, biologic agents, interleukins,interferons, cytokines, immuno-modulating agents, and monoclonalantibodies.

In one embodiment, an alkylating agent is used in combination withand/or added to the inventive compound/formulation. Examples ofalkylating agents include, but are not limited to bischloroethylamines(nitrogen mustards, e.g. chlorambucil, cyclophosphamide, ifosfamide,mechlorethamine, melphalan, uracil mustard), aziridines (e.g. thiotepa),alkyl alkone sulfonates (e.g. busulfan), nitrosoureas (e.g. carmustine,lomustine, streptozocin), nonclassic alkylating agents (altretamine,dacarbazine, and procarbazine), platinum compounds (carboplastin andcisplatin).

In another embodiment, cisplatin, carboplatin or cyclophosphamide isused in combination with and/or added to the inventivecompound/formulation.

In another embodiment, a member of the retinoids superfamily is used incombination with and/or added to the inventive compound/formulation.Retinoids are a family of structurally and functionally relatedmolecules that are derived or related to vitamin A (all-trans-retinol).Examples of retinoid include, but are not limited to, all-trans-retinol,all-trans-retinoic acid (tretinoin), 13-cis retinoic acid (isotretinoin)and 9-cis-retinoic acid.

In yet another embodiment, a hormonal agent is used in combination withand/or added to the inventive compound/formulation. Examples of such ahormonal agent are synthetic estrogens (e.g. diethylstibestrol),antiestrogens (e.g. tamoxifen, toremifene, fluoxymesterol andraloxifene), antiandrogens (bicalutamide, nilutamide, flutamide),aromatase inhibitors (e.g., aminoglutethimide, anastrozole andtetrazole), ketoconazole, goserelin acetate, leuprolide, megestrolacetate and mifepristone.

In yet another embodiment, a plant-derived agent is used in combinationwith and/or added to the inventive compound/formulation. Examples ofplant-derived agents include, but are not limited to, vinca alkaloids(e.g., vincristine, vinblastine, vindesine, vinzolidine andvinorelbine), camptothecin (20(S)-camptothecin,9-nitro-20(S)-camptothecin, and 9-amino-20(S)-camptothecin),podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)), andtaxanes (e.g., paclitaxel and docetaxel).

In yet another embodiment, a biologic agent is used in combination withand/or added to the inventive compound/formulation, such asimmuno-modulating proteins such as cytokines, monoclonal antibodiesagainst tumor antigens, tumor suppressor genes, and cancer vaccines.

Examples of interleukins that may be used in combination with and/oradded to the inventive compound/formulation include, but are not limitedto, interleukin 2 (IL-2), and interleukin 4 (IL-4), interleukin 12(IL-12). Examples of interferons that may be used in conjunction withdecitabine—glycerin formulations include, but are not limited to,interferon α, interferon β (fibroblast interferon) and interferon γ(fibroblast interferon). Examples of such cytokines include, but are notlimited to erythropoietin (epoietin □), granulocyte-CSF (filgrastim),and granulocyte, macrophage-CSF (sargramostim) Immuno-modulating agentsother than cytokines include, but are not limited to bacillusCalmette-Guerin, levamisole, and octreotide.

Example of monoclonal antibodies against tumor antigens that can be usedin conjunction with the inventive formulations include, but are notlimited to, HERCEPTIN® (Trastruzumab), RITUXAN® (Rituximab), MYLOTARG®(anti-CD33), and CAMPATH® (anti-CD52).

6. Indications for Compounds or Pharmaceutical Compositions of thePresent Invention

The pharmaceutical formulations according to the present invention maybe used to treat a wide variety of diseases that are sensitive to thetreatment with decitabine.

Preferable indications that may be treated using the pharmaceuticalformulations of the present invention include those involvingundesirable or uncontrolled cell proliferation. Such indications includebenign tumors, various types of cancers such as primary tumors and tumormetastasis, restenosis (e.g. coronary, carotid, and cerebral lesions),hematological disorders, abnormal stimulation of endothelial cells(atherosclerosis), insults to body tissue due to surgery, abnormal woundhealing, abnormal angiogenesis, diseases that produce fibrosis oftissue, repetitive motion disorders, disorders of tissues that are nothighly vascularized, and proliferative responses associated with organtransplants.

Generally, cells in a benign tumor retain their differentiated featuresand do not divide in a completely uncontrolled manner. A benign tumor isusually localized and nonmetastatic. Specific types benign tumors thatcan be treated using the present invention include hemangiomas,hepatocellular adenoma, cavernous haemangioma, focal nodularhyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bileduct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas,myxomas, nodular regenerative hyperplasia, trachomas and pyogenicgranulomas.

In a malignant tumor cells become undifferentiated, do not respond tothe body's growth control signals, and multiply in an uncontrolledmanner. The malignant tumor is invasive and capable of spreading todistant sites (metastasizing). Malignant tumors are generally dividedinto two categories: primary and secondary. Primary tumors arisedirectly from the tissue in which they are found. A secondary tumor, ormetastasis, is a tumor which is originated elsewhere in the body but hasnow spread to a distant organ. The common routes for metastasis aredirect growth into adjacent structures, spread through the vascular orlymphatic systems, and tracking along tissue planes and body spaces(peritoneal fluid, cerebrospinal fluid, etc.)

Specific types of cancers or malignant tumors, either primary orsecondary, that can be treated using this invention include breastcancer, skin cancer, bone cancer, prostate cancer, liver cancer, lungcancer, brain cancer, cancer of the larynx, gall bladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet celltumor, primary brain tumor, acute and chronic lymphocytic andgranulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullarycarcinoma, pheochromocytoma, mucosal neuronms, intestinalganglloneuromas, hyperplastic corneal nerve tumor, marfanoid habitustumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

Hematologic disorders include abnormal growth of blood cells which canlead to dysplastic changes in blood cells and hematologic malignanciessuch as various leukemias. Examples of hematologic disorders include butare not limited to acute myeloid leukemia, acute promyelocytic leukemia,acute lymphoblastic leukemia, chronic myelogenous leukemia, themyelodysplastic syndromes, and sickle cell anemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemiathat occurs in adults. Several inherited genetic disorders andimmunodeficiency states are associated with an increased risk of AML.These include disorders with defects in DNA stability, leading to randomchromosomal breakage, such as Bloom's syndrome, Fanconi's anemia,Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linkedagammaglobulinemia.

Acute promyelocytic leukemia (APML) represents a distinct subgroup ofAML. This subtype is characterized by promyelocytic blasts containingthe 15; 17 chromosomal translocation. This translocation leads to thegeneration of the fusion transcript comprised of the retinoic acidreceptor and a sequence PML.

Acute lymphoblastic leukemia (ALL) is a heterogenerous disease withdistinct clinical features displayed by various subtypes. Reoccurringcytogenetic abnormalities have been demonstrated in ALL. The most commoncytogenetic abnormality is the 9; 22 translocation. The resultantPhiladelphia chromosome represents poor prognosis of the patient.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferativedisorder of a pluripotent stem cell. CML is characterized by a specificchromosomal abnormality involving the translocation of chromosomes 9 and22, creating the Philadelphia chromosome. Ionizing radiation isassociated with the development of CML.

The myelodysplastic syndromes (MDS) are heterogeneous clonalhematopoietic stem cell disorders grouped together because of thepresence of dysplastic changes in one or more of the hematopoieticlineages including dysplastic changes in the myeloid, erythroid, andmegakaryocytic series. These changes result in cytopenias in one or moreof the three lineages. Patients afflicted with MDS typically developcomplications related to anemia, neutropenia (infections), orthrombocytopenia (bleeding). Generally, from about 10% to about 70% ofpatients with MDS develop acute leukemia.

Treatment of abnormal cell proliferation due to insults to body tissueduring surgery may be possible for a variety of surgical procedures,including joint surgery, bowel surgery, and cheloid scarring. Diseasesthat produce fibrotic tissue include emphysema. Repetitive motiondisorders that may be treated using the present invention include carpaltunnel syndrome. An example of cell proliferative disorders that may betreated using the invention is a bone tumor.

The proliferative responses associated with organ transplantation thatmay be treated using this invention include those proliferativeresponses contributing to potential organ rejections or associatedcomplications. Specifically, these proliferative responses may occurduring transplantation of the heart, lung, liver, kidney, and other bodyorgans or organ systems.

Abnormal angiogenesis that may be may be treated using this inventioninclude those abnormal angiogenesis accompanying rheumatoid arthritis,ischemic-reperfusion related brain edema and injury, cortical ischemic,ovarian hyperplasia and hypervascularity, (polycystic ovary syndrome),endometriosis, psoriasis, diabetic retinopaphy, and other ocularangiogenic diseases such as retinopathy of prematurity (retrolentalfibroplastic), muscular degeneration, corneal graft rejection,neuroscular glaucoma and Oster Webber syndrome.

Diseases associated with abnormal angiogenesis require or inducevascular growth. For example, corneal angiogenesis involves threephases: a pre-vascular latent period, active neovascularization, andvascular maturation and regression. The identity and mechanism ofvarious angiogenic factors, including elements of the inflammatoryresponse, such as leukocytes, platelets, cytokines, and eicosanoids, orunidentified plasma constituents have yet to be revealed.

In another embodiment, the pharmaceutical formulations of the presentinvention may be used for treating diseases associated with undesired orabnormal angiogenesis. The method comprises administering to a patientsuffering from undesired or abnormal angiogenesis the pharmaceuticalformulations of the present invention alone, or in combination withanti-neoplastic agent whose activity as an anti-neoplastic agent in vivois adversely affected by high levels of DNA methylation. The particulardosage of these agents required to inhibit angiogenesis and/orangiogenic diseases may depend on the severity of the condition, theroute of administration, and related factors that can be decided by theattending physician. Generally, accepted and effective daily doses arethe amount sufficient to effectively inhibit angiogenesis and/orangiogenic diseases.

According to this embodiment, the pharmaceutical formulations of thepresent invention may be used to treat a variety of diseases associatedwith undesirable angiogenesis such as retinal/choroidalneuvascularization and corneal neovascularization. Examples ofretinal/choroidal neuvascularization include, but are not limited to,Bests diseases, myopia, optic pits, Stargarts diseases, Pagets disease,vein occlusion, artery occlusion, sickle cell anemia, sarcoid, syphilis,pseudoxanthoma elasticum carotid abostructive diseases, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, diabeticretinopathy, macular degeneration, Bechets diseases, infections causinga retinitis or chroiditis, presumed ocular histoplasmosis, parsplanitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications, diseases associatedwith rubesis (neovascularization of the angle) and diseases caused bythe abnormal proliferation of fibrovascular or fibrous tissue includingall forms of proliferative vitreoretinopathy. Examples of cornealneuvascularization include, but are not limited to, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, diabetic retinopathy,retinopathy of prematurity, corneal graft rejection, Mooren ulcer,Terrien's marginal degeneration, marginal keratolysis, polyarteritis,Wegener sarcoidosis, Scleritis, periphigoid radial keratotomy,neovascular glaucoma and retrolental fibroplasia, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections and Kaposi sarcoma.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used for treating chronic inflammatory diseasesassociated with abnormal angiogenesis. The method comprisesadministering to a patient suffering from a chronic inflammatory diseaseassociated with abnormal angiogenesis the pharmaceutical formulations ofthe present invention alone, or in combination with an anti-neoplasticagent whose activity as an anti-neoplastic agent in vivo is adverselyaffected by high levels of DNA methylation. The chronic inflammationdepends on continuous formation of capillary sprouts to maintain aninflux of inflammatory cells. The influx and presence of theinflammatory cells produce granulomas and thus, maintains the chronicinflammatory state. Inhibition of angiogenesis using the pharmaceuticalformulations of the present invention may prevent the formation of thegranulosmas, thereby alleviating the disease. Examples of chronicinflammatory disease include, but are not limited to, inflammatory boweldiseases such as Crohn's disease and ulcerative colitis, psoriasis,sarcoidosis, and rheumatoid arthritis.

Inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis are characterized by chronic inflammation and angiogenesis atvarious sites in the gastrointestinal tract. For example, Crohn'sdisease occurs as a chronic transmural inflammatory disease that mostcommonly affects the distal ileum and colon but may also occur in anypart of the gastrointestinal tract from the mouth to the anus andperianal area. Patients with Crohn's disease generally have chronicdiarrhea associated with abdominal pain, fever, anorexia, weight lossand abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea. Theseinflammatory bowel diseases are generally caused by chronicgranulomatous inflammation throughout the gastrointestinal tract,involving new capillary sprouts surrounded by a cylinder of inflammatorycells. Inhibition of angiogenesis by the pharmaceutical formulations ofthe present invention should inhibit the formation of the sprouts andprevent the formation of granulomas. The inflammatory bowel diseasesalso exhibit extra intestinal manifectations, such as skin lesions. Suchlesions are characterized by inflammation and angiogenesis and can occurat many sites other the gastrointestinal tract. Inhibition ofangiogenesis by the pharmaceutical formulations of the present inventionshould reduce the influx of inflammatory cells and prevent the lesionformation.

Sarcoidosis, another chronic inflammatory disease, is characterized as amulti-system granulomatous disorder. The granulomas of this disease canform anywhere in the body and, thus, the symptoms depend on the site ofthe granulomas and whether the disease is active. The granulomas arecreated by the angiogenic capillary sprouts providing a constant supplyof inflammatory cells. By using the pharmaceutical formulations of thepresent invention to inhibit angionesis, such granulomas formation canbe inhibited. Psoriasis, also a chronic and recurrent inflammatorydisease, is characterized by papules and plaques of various sizes.Treatment using the pharmaceutical formulations of the present inventionshould prevent the formation of new blood vessels necessary to maintainthe characteristic lesions and provide the patient relief from thesymptoms.

Rheumatoid arthritis (RA) is also a chronic inflammatory diseasecharacterized by non-specific inflammation of the peripheral joints. Itis believed that the blood vessels in the synovial lining of the jointsundergo angiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Treatment using thepharmaceutical formulations of the present invention alone or inconjunction with other anti-RA agents may prevent the formation of newblood vessels necessary to maintain the chronic inflammation and providethe RA patient relief from the symptoms.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used for treating diseases associated withabnormal hemoglobin synthesis. The method comprises administering thepharmaceutical formulations of the present invention to a patientsuffering from disease associated with abnormal hemoglobin synthesis.Decitabine containing formulations stimulate fetal hemoglobin synthesisbecause the mechanism of incorporation into DNA is associated with DNAhypomethylation. Examples of diseases associated with abnormalhemoglobin synthesis include, but are not limited to, sickle cell anemiaand β-thalassemia.

In yet another embodiment, the pharmaceutical formulations of thepresent invention may be used to control intracellular gene expression.The method comprises administering the pharmaceutical formulations ofthe present invention to a patient suffering from disease associatedwith abnormal levels of gene expression. DNA methylation is associatedwith the control of gene expression. Specifically, methylation in ornear promoters inhibit transcription while demethylation restoresexpression. Examples of the possible applications of the describedmechanisms include, but are not limited to, therapeutically modulatedgrowth inhibition, induction of apoptosis, and cell differentiation.

Gene activation facilitated by the pharmaceutical formulations of thepresent invention may induce differentiation of cells for therapeuticpurposes. Cellular differentiation is induced through the mechanism ofhypomethylation. Examples of morphological and functionaldifferentiation include, but are not limited to differentiation towardsformation of muscle cells, myotubes, cells of erythroid and lymphoidlineages.

Although exemplary embodiments of the present invention have beendescribed and depicted, it will be apparent to the artisan of ordinaryskill that a number of changes, modifications, or alterations to theinvention as described herein may be made, none of which depart from thespirit of the present invention. All such changes, modifications, andalterations should therefore be seen as within the scope of the presentinvention.

Examples 1. Synthesis of Phosphoramidite Building Blocks and 3′-O-CappedDerivatives

The present invention also provides effective chemical methods forsynthesis of the following novel phosphoramidite building blocks (FIG.2A).

The 4-amine functional group of 1a can be protected via transformationinto various protective groups (R₁), such as carbamates with methyl,ethyl, 9-fluorenylmethyl, 9-(2-sulfo)fluorenylmethyl,9-(2,7-dibromo)fluorenylmethyl, 17-tetrabenzo[a,c,g,i]fluorenylmethyl,2-chloro-3-indenylmethyl, benz[f]inden-3-ylmethyl,2,7-di-tert-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)methyl,1,1-dioxobenzo[1)]thiophene-2-ylmethyl, 2,2,2-trichloroethyl,2-trimethylsilylethyl, 2-phenylethyl, 1-(1-adamantyl)-1-methylethyl,2-chloroethyl, 1,1,-dimethyl-2-haloethyl, 1,1-dimethyl-2,2-dibromethyl,1,1-dimethyl-2,2,2-trichlroethyl, 1-methyl-1-(4-biphenylyl)ethyl,1-(3,5-di-tert-butylphenyl)-1-methylethyl, 2-(2′- and 4′-pyridyl)ethyl,2,2-bis(4′-nitrophenyl)ethyl, N-(2-pivaloylamino)-1,1-dimethylethyl,2-[(2-nitrophenyl)dithio]-1-phenylethyl,2-(N,N-dicyclohexylcarboxamido)ethyl, t-butyl, 1-adamantyl, 2-adamantyl,vinyl, allyl, 1-isopropylallyl, cinnayl, 4-nitrocinnamyl,3-(3′-pyridyl)prop-2-enyl, 8-quinolyl, N-hydroxypiperidinyl,alkyldithio, benzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl,p-chlorobenzyl, 2,4-dichlorobenzyl, 4-methylsulfinylbenzyl,9-anthrylmethyl, diphenylmethyl, 2-methylthioethyl,2-methylsulfonylethyl, 2-(p-toluenesulfonyl)ethyl,[2-(1,3-dithianyl)]methyl, 4-methylthiphenyl, 2,4-dimethylthiphenyl,2-phosphonioethyl, 1-methyl-1-(triphenylphosphonio)ethyl,1,1-dimethyl-2-cyanoethyl, 2-dansylethyl, 4-phenylacetoxybenzyl,4-azidobenzyl, 4-azidomethoxybenzyl, m-chloro-p-acyloxybenzyl,p-(dihydroxyboryl)benzyl, 5-benzisoxazolylmethyl,2-(trifluoroethyl)-6-chromonylmethyl, m-nitrophenyl,3,5-dimethoxybenzyl, 1,-methyl-1-(3,5-dimethoxyphenyl)ethyl,α-methylnitropiperonyl, o-nitrophenyl, 3,4-dimethoxy-6-nitrobenzyl,phenyl(o-nitrophenyl)ethyl, 2-(2-nitrophenyl)ethyl, 6-nitroveratryl,4-methoxyphenacyl, 3′, 5′-dimethoxybenzoin, t-amyl, S-benzylthio,butynyl, p-cyanobenzyl, cyclohexyl, cyclopentyl, cyclopropylmethyl,p-decyloxybenzyl, diisopropylmethyl, 2,2-dimethoxycarbonylvinyl,o-(N,N-dimethylcarboxamido)benzyl,1,1-dimethyl-3-(N,N-dimethycarboxamido)propyl, 1,1-dimethylpropynyl,2-furanylmethyl, 2-iodoethyl, isobornyl, isobutyl, isonicotinyl,p-(p′-methoxyphenylazo)benzyl, 1-methylcyclobutyl, 1-methylcyclohexyl,1-methyl-1-cyclopropylmethyl, 1-methyl-1-(p-phenylazophenyl)ethyl,1-methyl-1-phenylethyl, 1-methyl-1-(4′-pyridyl)ethyl, phenyl,p-(phenylazo)benzyl, 2,4,6-tri-t-butylphenyl,4-(trimethylammonium)benzyl, 2,4,6-trimethylbenzyl; ureas withphenothiazinyl-(10)-carbonyl, N′-p-toluenesulfonylaminocarbonyl,N′-phenylaminothiocarbonyl; amides such as formamide, acetamide,phenoxyacetamide, trichloroacetamide, trifluoroacetamide,phenyacetamide, 3-phenylpropamide, pent-4-enamide,o-nitrophenylacetamide, o-nitrophenoxyacetamide,3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide,3-(4-t-butyl-2,6-dinitrophenyl)-2,2-dimethylpropanamide,o-(benzoyloxymethyl)benzamide,2-[t-butyldiphenylsiloxy)methyl)methyl]benzamide,3-(3′,6′-dioxo-2′,4′,5′-trmethylcyclohexa-1′,4′-diene)-3,3-dimethylpropionamide,o-hydroxy-trans-cinnamide, acetoacetamide, p-toluenesulfonamide, andbenzesulfonamide. The 4-O-methoxy (1b) and 4-S-methylthio (1c) analogsof decitabine can be obtained by modifying the published procedure fordecitabine synthesis by separating the α and β anomers of1-(2-deoxy-3,5-di-O-p-chlorobenzoyl orbenzoyl-D-ribofuranosyl)-4-methoxy or methylthio-1,3,5-triazin-2(H)-oneand removing the 3,5-protective groups without treatment with methanolicammonia. Pliml and Sorm (1964) Collect. Czech. Chem. Commun. 29:2576-2577; Piskala and Sorm (1978) Nucleic Acid Chemistry (by Towsendand Tipson, Wiley, 1978), pp. 443-449.

Protection of the 5′-OH is achieved by dissolving the 4-amino protecteddecitabine and 4-methoxy and 4-methylthio analogs (1b, 1c) in anhydrouspyridine (5 mL/mmol) before adding dimethoxytrityl chloride (1.1equivalents).

For example, decitabine (1.2 g) was twice co-distilled with anhydrouspyridine and dissolved in 20 ml dry DMF. Hexamethyldisilazane (2.8 mL)was added. The solution was stirred and left overnight. The solvent wasevaporated in vacuo, and the remaining residue was dissolved in tolueneand evaporated twice. The 3′, 5′-di trimethylsilyl5-aza-2′-deoxycytidine (R_(f)=0.67, 4:1 dichloromethane/methanol) wasco-distilled twice with dry pyridine (˜10 mL) and dissolved in drypyridine (20 mL). Phenoxyacetic anhydride (1.5 g) was added, and theresulting solution was stirred for 1 hour. A further 0.18 gphenoxyacetic anhydride (0.18 g) was added and stirred for another hour.The reaction mixture was evaporated in vacuo to dryness and co-distilled(3×) with toluene. The residue was dissolved in dichloromethane (˜50 mL)and extracted with 1M aqueous NaHCO₃ solution (˜50 mL), which wasre-extracted with dichloromethane (˜20 mL). The combined organic phaseswere dried over sodium sulfate and reduced in vacuo to yield crude 3′,5′-di trimethylsilyl-N-phenoxyacetyl 5-aza-2′-deoxycytidine (3 g;R_(f)=0.82, 9:1 dichloromethane/methanol). The crude material wasdissolved in anhydrous DMF (20 mL) and transferred to a 50 mL plasticfalcon tube, and TAS-F (2.4 g) was added (gas evolved). The reactionproceeded for 4 hours at 22° C. (the vial was not fully closed to reducepressure built up). The DMF was evaporated in vacuo and the remainingresidue was subjected to column chromatography (30 g silica gel, 2.5 cmcolumn, 99:1 to 9:1 dichloromethane/methanol). A white solidN-phenoxyacetyl 5-aza-2′-deoxycytidine (0.81 g; R_(f)=0.26, 9:1dichloromethane/methanol) was obtained. This compound (0.6 g) was twiceco-distilled with anhydrous pyridine and dissolved in anhydrous pyridine(20 mL) before dimethoxytrityl chloride (0.9 g) was added and stirredfor 2 hours at 22° C. Solvents were removed in vacuo and co-distilled(3×) with toluene. The residue was dissolved in dichloromethane (50 mL)and extracted with 1M aqueous NaHCO₃ solution (˜50 mL), which wasre-extracted with dichloromethane (˜20 mL). The combined organic phaseswere dried over sodium sulfate and reduced in vacuo. The residue wassubjected to silica gel chromatography (dichloromethane-100% to 95:5dichloromethane/methanol), which yielded5′-dimethoxytrityl-N-phenoxyacetyl 5-aza-2′-deoxycytidine (0.35 g, 0.53mmole, 32%; R_(f)=0.49, 9:1 dichloromethane/methanol). This intermediate(0.3 g) was dissolved in dry acetonitrile (2 mL) before 0.3 Mbenzylthiotetrazole (0.9 mL) solution in dry acetonitrile andcyanoethyltetraisopropyl phosphorodiamidite (0.17 mL) were added. Themixture was stirred at 22° C. for 1.5 hours. TLC (2:1 ethylacetate/hexanes+2% TEA) showed a diastereoisomeric mixture withR_(f)=0.27 and 0.36. Solvent was removed in vacuo and the remainingresidue subjected to column chromatography (20 g silica gel, 2.5 cmcolumn, 9:1 hexanes/ethyl acetate+2% TEA (300 mL), 1:1 hexanes/ethylacetate+1% TEA (200 mL), 1:2 hexanes/ethyl acetate+0% TEA (250 mL). Thedecitabine phosphoramidite building 1d, where R₁=phenoxyacetyl (0.297 g,0.34 mmol, 76%) eluted with the 1:2 hexanes/ethyl acetate. ESI-MS of 1d(calculated exact mass for C₄₆H₅₃N₆O₉P is 864.36) exhibited m/z 864.1and 966.4 [M+NEt₃+H⁺]⁺; ³¹P NMR (CDCl₃, 500 MHz) exhibited 149.17 and149.0 ppm; ¹H NMR (CDCl₃, 500 MHz) exhibited chemical shifts (ppm) 8.63& 8.59 (1H, doublet, H-6), 7.4-6.6 (18H, multiplet, aromatic DMTr/Pac),6.05 (1H, triplet, H-1′), 4.79 (2H, singlet, CH₂ of Pac), 4.59 (1H,singlet, H-4′), 4.25 to 4.20 (1H, doublet, H-3′), 3.8-3.7 (1H,multiplet, P—O—CH₂), 3.70 (3H, singlet, CH₃O of DMTr), 3.68 (3H,singlet, CH₃O of DMTr), 3.6-3.48 (3H, multiplet, two CH's of isopropyland one P—O—CH₂), 3.36-3.27 (2H, multiplet, H-5′), 2.80 (1H, singlet,H-2′), 2.53 (1H, singlet, H-2′), 2.40 (2H, multiplet, CH₂CN), 1.1 (12H,CH₃ of isopropyl).

In addition, minor modification of published procedures allow access to3′- and 5′-O-capped derivatives (FIG. 3A, 1g, 1h, 1i, 1j, 1k, 1l)(Bagnall, Bell and Pearson (1978) J. of Fluorine Chem. 11: 93-107),where the cap can be alkyl groups, esters and fatty acid esters, glycolderivatives such as ethylene and propylene glycols; and protecteddecitabine 3′-linked onto controlled-pore glass support (FIG. 3B, 1m,1n, 1o). Alul, Singman, Than and Letsinger (1991) 19: 1527-1532.

Other decitabine derivatives have the 3′-OH protected with esters (whichinclude but are not limited to acetyl, benzoyl, and halobenzoyl; andfatty acids) and ethers (which include but are not limited top-nitrophenylethyl, methoxymethyl, methylthiomethyl,(phenyldimethylsilyl)methoxymethyl, benzyloxymethyl,p-methoxybenzyloxymethyl, p-nitrobenzyloxymethyl,o-nitrobenzyloxymethyl, (4-methoxyphenoxy)methyl, t-butoxymethyl,4-pentenyloxymethyl, siloxymethyl, 2-methoxyethoxymethyl,2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,2-(trimethylsilyl)ethoxymethyl, menthoxymethyl, tetrahydropyranyl,tetrahydrofuranyl, and 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl),glycol derivatives such as ethylene and propylene glycol, as shown inFIG. 4.

2. Synthesis of DpG and GpD Dinucleotides and Tetranucleotides on SolidSupport

The DpG and GpD dinucleotides and tetranucleotides can be synthesized bystandard procedures (FIG. 5) with slight modification for increasedcoupling times (>2 minutes). Beaucage and Caruthers (1981) Tet. Lett.22: 1859-1862; McBride and Caruthers (1983) Tet. Lett. 24: 245-248.Synthesis of GpD dinucleotide 2a and DpGpGpD tetranucleotide 3a can beinitiated with the coupling of 1m, 1n or 1o with similarly basedprotected 5′-O-DMTr2′-deoxyguanosine-3′-O-cyanoethyl-N,N-diisopropylphosphoramidite and5′-O-DMTr2′-deoxy-5-aza-cytidine-3′-O-cyanoethyl-N,N-diisopropylphosphoramidites(1d, 1e or 1f), as shown in FIG. 6A. Subsequent release from the solidsupport (such as controlled pore glass, CPG) and removal of carbamateprotective groups with bases such as DBU/pyridine (or acetonitrile), andmethanolic ammonia for removal of the 4-O-methoxy and 4-O-methylthioprotective groups, yield the desired oligonucleotides with the last DMTrgroup on or off. DpGpGpD (3b) can be similarly obtained (FIG. 7).

For example (FIGS. 6A and 6B), an Amersham ÄKTA Oligopilot 10 system isloaded with a protected decitabine-linked CPG solid support 1m (whereR₁=phenoxyacetyl), which is coupled with 2-2.5 equivalents of tert-butylphenoxyacetyl 2′-deoxyguanosine phosphoramidite in presence of 60% of0.3 M benzylthiotetrazole activator (in acetonitrile) for 2.5 minutes.The CPG solid support containing protected GpD dinucleotide is treatedwith 20 mL of 50 mM K₂CO₃ in methanol for 1 hour and 20 minutes. Thecoupled product is oxidized with 2 M tert-butylhydroperoxide in dryacetonitrile (prepared by dissolving tert-butylhydroperoxide in 80%tert-butylperoxide) for 5 minutes. The dimethoxy trityl protective groupis removed with 3% dichloroacetic acid in toluene. The CPG solid supportis washed with dry methanol; the filtrate is neutralized by addition of2 mL of 1 M acetic acid in methanol. The solution is concentrated byrotary evaporation; the residue is taken up in 200 mM triethylammoniumacetate (pH 6.9), washed with acetonitrile (500 μL of 50% aqueousacetonitrile), and filtered through a syringe filter. The GpDdinucleotide is subsequently purified by the ÄKTA Explorer 100 HPLC witha Gemini C18 preparative column (Phenomenex), 250×21.2 mm, 10 μm withguard column (Phenomenex), 50×21.2 mm, 10 μm, with 50 mMtriethylammonium acetate (pH 7) in MilliQ water (Mobile Phase A) and 80%acetonitrile in MilliQ water (Mobile Phase B), with 2% to 20/25% MobilePhase B in column volumes. The ESI-MS (−ve) of GpD dinucleotide 2a,where X⁺=triethylammonium (calculated exact mass for the neutralcompound C₁₈H₂₄N₉O₁₀P is 557.14), exhibited m/z 556.1 [M-H]⁻ and 1113.1for [2M-H]⁻ (see mass spectrum in FIG. 30).

When the cycle is repeated three times with 2-2.5 equivalents oftert-butyl phenoxyacetyl 2′-deoxyguanosine or phenoxyacetyl5-aza-2′-deoxycytidine phosphoramidite in presence of 60% of 0.3 Mbenzylthiotetrazole activator (in acetonitrile) for 2.5 minutes and 10minutes, respectively, (FIGS. 6b and 7), the DpGpGpD tetranucleotide 3bis obtained, where X⁺=triethylammonium (calculated exact mass for theneutral compound C₃₆H₄₇N₁₈O₂₂P₃ is 1176.23), exhibited m/z 587.2 for[M-2H]²⁻ and 1175.2 [M-H]⁻ (FIG. 32).

In addition, DpG dinucleotide 2b and GpDpGpD 3c can be synthesized bycoupling is (where R₁=carbamate protective group) with phosphoramiditebuilding blocks 1d, 1e or 1f (FIG. 8). GpDpDpG and GpDpG (3c′) canlikewise be obtained (FIG. 9).

For example, when a protected 2′-deoxyguanosine-linked CPG solid support1s (where R₁=tert-butyl phenoxyacetyl), which is coupled with 2-2.5equivalents of phenoxyacetyl decitabine phosphoramidite (FIG. 2A, 1d,where R₁=phenoxyacetyl; see mass spectrum in FIG. 38) in the presence of60% of 0.3 M benzylthiotetrazole activator (in acetonitrile) for 10minutes. The CPG solid support containing protected DpG dinucleotide istreated with 20 mL of 50 mM K₂CO₃ in methanol for 1 hour and 20 minutes.The coupled product is oxidized, protective group removed, washed,filtered, and purified as described for GpD dinucleotide. The ESI-MS(−ve) of DpG dinucleotide 2b, where X⁺=triethylammonium (calculatedexact mass for the neutral compound C₁₈H₂₄N₉O₁₀P is 557.14), exhibitedm/z 556.1 [M-H]⁻ and 1113.1 for [2M-H]⁻ (see mass spectrum in FIG. 31).The DpG dinucleotide 2b, where X⁺=sodium, is obtained by re-dissolvingthe triethylammonium salt in 4 ml water, 0.2 ml 2M NaClO₄ solution. When36 mL acetone is added, the dinucleotide precipitates. The solution iskept at −20° C. for several hours and centrifugated at 4000 rpm for 20minutes. The supernatant is discarded and the solid is washed with 30 mLacetone followed by an additional centrifugation at 4000 rpm for 20minutes. The precipitate is dissolved in water and freeze dried, whichexhibited m/z 556.0 [M-H]⁻ (see mass spectrum in FIG. 36).

When the cycle is repeated twice with 2-2.5 equivalents of tert-butylphenoxyacetyl 2′-deoxyguanosine or phenoxyacetyl 5-aza-2′-deoxycytidinephosphoramidite in presence of 60% of 0.3 M benzylthiotetrazoleactivator (in acetonitrile) for 2.5 minutes and 10 minutes,respectively, the GpDpG trinucleotide 3c′ is obtained, whereX⁺=triethylammonium (calculated exact mass for the neutral compoundC₂₈H₃₆N₁₄O₁₆P₂ is 886.2), which exhibited m/z 885.16 [M-H]⁻ (see massspectrum FIG. 33).

When cycle is repeated three times, the DpGpDpG tetranucleotide 3c isobtained, where X⁺=triethylammonium (calculated exact mass for theneutral compound C₃₆H₄₇N₁₈O₂₂P₃ is 1176.23), which exhibited m/z 587.4for [M-2H]²⁻ and 1175.4 [M-H]⁺ (see mass spectrum in FIG. 34).

When the phosphite triester, newly formed during the coupling step, isconverted to the corresponding phosphorothioate triester with 5%phenylacetyl disulfide (PADS) in dichloroethane/sym collidine 4/1 (v/v),4.3 mL solution (3.6 column volumes), flow rate 50 cm/h (contact time 3column volumes), the phosphorothioate derivative of 2b can be obtained.The sulfurization is completed within 3 minutes, at which time excessreagent is removed from the reaction vessel by washing withacetonitrile. Subsequent deprotection and purification, as described for2a, gives phosphorothioate DpG (Sp & Rp, FIG. 13, 2e), whereX⁺=triethylammonium (calculated exact mass for the neutral compoundC₁₈H₂₄N₉O₉PS is 573.12), which exhibited m/z 571.9 for [M-H]⁺ (see massspectrum in FIG. 35).

When cycle is repeated once with DMT hexaethylenglycol phosphoramidite(60% activator, 7 min coupling time), followed by standard oxidation andpurification as described for 2a, the HEG-DpG dinucleotide 2d isobtained (FIG. 12), where X⁺=triethylammonium and Cap=hexaethyleneglycolphosphate (calculated exact mass for the neutral compound C₃₀H₄₉N₉₈O₁₉P₂is 901.71), which exhibited m/z 900.4 [M-H]⁺ (see mass spectrum in FIG.37).

3. Inhibition of DNA Methylation by DpG and GpD Di-, Tri- andTetranucleotides

The demethylating activity of DpG and GpD di-, tri-, andtetranucleotides were tested in a cell-based GFP (green fluorescentprotein) assay. This assay, which is schematically illustrated in FIG.25, has a GFP gene regulated by the CMV promoter and is sensitive to themethylation of CpG sites within the promoter. A decrease in methylationresulting from exposure to a methylation inhibitor leads to GFPexpression and is readily scored. Specifically, the CMV-EE210 cell linecontaining the epigenetically silenced GFP transgene was used to assayfor reactivation of GFP expression by flow cytometry. CMV-EE210 was madeby transfecting NIH 3T3 cells with the pTR-UF/UF1/UF2 plasmid (Zolotuhinet al., 1996), which is comprised of pBS(+) (Stratagene, Inc.)containing a cytomegalovirus (CMV) promoter driving a humanized GFP geneadapted for expression in mammalian cells. After transfection,high-level GFP expressing cells were initially selected by FACS analysisand sorting using a MoFlo cytometer (Cytomation, Inc.). Decitabine,potent inhibitor of mammalian DNMT1, was used as a positive control. Toscreen for reactivation of CMV-EE210, decitabine (at 1 μM) or a testcompound (at a concentration of 30-50 μM) was added to complete medium(phenol red free DMEM (Gibco, Life Technologies) supplemented with 10%fetal bovine serum (Hyclone)). Cells were then seeded to 30% confluence(˜5000 cell/well) in 96 well plate containing the test compounds andgrown for three days in at 37° C. in 5% CO₂. The plates were examinedunder a fluorescent microscope using a 450-490 excitation filter (I3filter cube, Leica, Deerfield Ill.). Wells were scored g1 positive if(10%) of viable cells express GFP, g2 positive if 30% of viable cellsexpress GFP and g3 if >75% of the viable cells express GFP. GFP 50 isthe concentration of an inhibitor that (like an IC₅₀) is the dose atwhich the GFP expression level goes from g3 to g1/2. Table 1 lists theresults of the test for decitabine, DpG, GpD, GpDpG, DpGpGpD and DpGpDpGas DNA methylation inhibitors. As shown in Table 1, all of the 5oligonucleotide analogues tested were able to inhibit DNA methylationeffectively at low concentrations, resulting in reactivation of thetranscription of the GFP gene.

TABLE 1 Preliminary screening of demethylating activity GFP ExpressionIC 50 Compound Level (nM) Decitabine g3 500 DpG g3 400 GpD g3 700 GpDpGg3 1800 DpGpGpD g3 1100 DpGpDpG g3 1400

4. Synthesis of DpG and GpD Dinucleotides and Tetranucleotides inSolution

For the synthesis of these oligonucleotides in large scale, the use ofsoluble polymeric supports is desirable. Bayer and Mutter, (1972) Nature237: 512-513; Bonora (1995) Appl. Biochem. Biotechnol. 54: 3-17. Thepolymer support poly (ethylene glycol) or PEG allows synthetic processto be carried out in a homogeneous phase and assures an easyintermediate purification step through simpleprecipitation-and-filtration procedures. Harris Poly(ethylene glycol)Chemistry. Biotechnical and Biomedical Applications, J. M. Harris (Ed.).Plenum Press, New York (USA), 1992, pp. 1-14 [book citation]. Forexample, 3′-linked derivatives such as 1t, 1u, or 1v (FIG. 10) can beeasily adapted to the standard phosphoramidite-based chemistry employedin the preceding solid-phase procedures to give the DpG and GpD di- andtetranucleotides 2a, 2b, 3a, 3b, 3c, 3d.

Alternatively, the novel DpG dinucleotide 2a can be prepared in solutionby coupling derivatives 1p, 1q, or 1r with similarly based protected5′-O-DMTr2′-deoxyguanosine-3′-O-cyanoethyl-N,N-diisopropylphosphoramidite and GpDfrom the coupling of similarly 3′-protected 2′-deoxyguanosine with5′-O-DMTr2′-deoxy-5-aza-cytidine-3′-O-cyanoethyl-N,N-diisopropylphosphoramidites(1d, 1e or 1f) in acetonitrile and/or dichloromethane, followed byoxidation with iodine/water, deprotection of the base protective groups,and removal of the DMTr group (as in the standard cycle foroligonucleotide synthesis).

In addition, the novel DpG (2c) dinucleotide with the terminal 3′-OH and5′-OH capped with methyl group can be prepared by coupling 3′-O-methylderivative 1g, 1h or 1i with a 5′-O-methyl derivative of2′-deoxyguanosine-3′-O-cyanoethyl-N,N-diisopropylphosphoramidite 1w(FIG. 11), followed by oxidation with iodine/water, deprotection of thebase protective groups, and removal of the DMTr group (as in thestandard cycle for oligonucleotide synthesis). Dinucleotide GpD (2d) canlikewise be prepared by coupling 3′-O-methyl 2′-deoxyguanosinederivative 1× with 5′-O-methyl derivative of2′-5-azacytidine-3′-O-cyanoethyl-N,N-diisopropylphosphoramidite 1j, 1kor 1l (FIG. 12).

5. Synthesis of DpG and GpD Oligonucleotides Resistant to CytidineDeaminases and Nucleases

In general, oligonucleotides in biological fluids are subject tonuclease degradation. Stein and Cheng (1993) Science 261: 1004-1012;Cohen (1994) Adv. Pharmacol. 25: 319-339. To increase stability andresistance to nuclease degradation phosphothioate dinucleotide andtetranucleotide derivatives such as 2e, 2f, 3e, 3f, 3g, and 3h (FIGS.13, 14 and 15) are also made, where the internucleotide non-bridgingoxygen is replaced with sulfur. Standard phosphoramidite protocols areused, except for the substitution of bis(O,O-diisopropoxyphosphinothioyl) disulfide (S-tetra) for iodine during the oxidationstep. Zon and Stec (1991) In Eckstein, F. (ed.), ‘PhosphorothioateAnalogues’ in Oligonucleotides and Their Analogs: A Practical Approach.IRL Press, pp. 87-108; Zon, G. (1990) In Hancock, W. S. (ed.), HighPerformance Liquid Chromatography in Biotechnology. Wiley, New York, Ch.14, pp. 310-397 [book citations]; Stec, Uznanski, Wilk, Hirschbein,Fearon, and Bergot (1993) Tet. Lett. 34: 5317-5320; Iyer, Phillips,Egan, Regan, and Beaucage (1990) J. Org. Chem. 55: 4693-4699.

Another potential hindrance to the application of these oligonucleotidesas pharmaceuticals is the ubiquitous presence of cytidine deaminase(CDA) since deamination of decitabine results in total loss of activity.Momparler, Cote and Eliopoulos (1997) Leukemia 11 (Suppl.1): 1-6;Chabot, Bouchard and Momparler (1983) Biochem. Pharmacol. 32: 1327-1328;Laliberte, Marquez and Momparler (1992) Cancer Chemother. Pharmacol. 30:7-11. To address this problem, the oligonucleotides containingdecitabine derivatives with the 4-NH₂ is replaced by 4-NR₃R₄ (where R₃and R₄ can be alkyl, alkyl amine, and alkyl alcohol) are also preparedto give derivatives such as 2g, 2h, 2i, 2j, 2k, 2l, 2m, 2n, 3i, 3j, 3k,3l, 3m, 3n, 3o, and 3p (FIGS. 16, 17, 18, 19, 20 and 21). Standardphosphoramidite protocols are used, except for the substitution of alkylamines, alkyl diamines, and hydroxyl amines for ammonia in methanolduring the removal of 4-methoxy and 4-methylthio. Since secondary andtertiary amines, diamines, and hydroxyl amines make worse leaving groupsthan ammonia, these derivatives are more difficult to deaminate.

6. Synthesis of DpG and GpD-Rich Oligonucleotides Based on the CpGIslands of the Promoter Regions of Cancer Related Genes Such as P15(CDKN2B), BRCA1, and P16 (CDKN2A)

Oligonucleotide analogues rich in DpG and GpD islets that range inlength from 5 to 100 bases can be prepared, where D can be decitabine ordecitabine analogues. Unlike the above described DpG and GpDdinucleotides and tetranucleotides, these relatively longer DpG andGpD-rich oligonucleotide analogues not only function restrictivelywithin the CpG islands of the promoter regions but specific to a segmentwithin the promoter region sequence for cancer related genes such as P15(CDKN2B), P16 (CDKN2A) and BRAC1. For examples, 8-mer, 10-mer, and12-mer DpG and GpD-rich oligonucleotide analogues (FIG. 26) based on theP15, P16, and BRCA1 promoter region sequences (FIGS. 27, 28 and 29,respectively) can be prepared by using phosphoramidite building block1d, 1e or 1f in a standard solid phase oligonucleotide synthesis. Moreexamples of oligonucleotides that can be modified to incorporate5-aza-cytosine therein are listed in FIGS. 27, 28 and 29. Theseoligonucleotide analogues can function like primers and get incorporatedinto replicating DNA only at that specific segment of the promoterregion sequence of P15, P16 or BRAC1, thus effectively and selectivelyinhibiting methylation of the promoter region.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The invention claimed is:
 1. A compound of the formula:

wherein X⁺ is Na⁺.
 2. A pharmaceutical composition comprising a compoundof the formula:

wherein X⁺ is Na⁺; and a pharmaceutically-acceptable carrier.
 3. Thepharmaceutical composition of claim 2, wherein thepharmaceutically-acceptable carrier is an emulsion comprising a cationicemulsifier.
 4. The pharmaceutical composition of claim 2, wherein thecompound is in solid form.
 5. The pharmaceutical composition of claim 2,wherein the pharmaceutically-acceptable carrier is a solution containingless than 40% water.
 6. The pharmaceutical composition of claim 2,wherein the pharmaceutically-acceptable carrier is a solution containingless than 5% water.
 7. The pharmaceutical composition of claim 2,wherein the pharmaceutically-acceptable carrier is a solution containingless than 1% water.
 8. The pharmaceutical composition of claim 2,wherein the pharmaceutically-acceptable carrier is substantiallyanhydrous.
 9. The pharmaceutical composition of claim 2, wherein thepharmaceutically-acceptable carrier is a solution comprising a solventselected from the group consisting of ethanol, glycerin, propyleneglycol, polyethylene glycol, and combinations thereof.
 10. Thepharmaceutical composition of claim 2, wherein thepharmaceutically-acceptable carrier is a solution containing less than5% water comprising a solvent selected from the group consisting ofethanol, glycerin, propylene glycol, polyethylene glycol, andcombinations thereof.
 11. The pharmaceutical composition of claim 2,wherein the pharmaceutically-acceptable carrier comprises a combinationof propylene glycol and glycerin.
 12. The pharmaceutical composition ofclaim 11, wherein the concentration of propylene glycol in thepharmaceutically-acceptable carrier is between 10% and 80%.
 13. Thepharmaceutical composition of claim 11, wherein the concentration ofpropylene glycol in the pharmaceutically-acceptable carrier is between50% and 70%.
 14. The pharmaceutical composition of claim 2, formulatedfor administration by injection.
 15. The pharmaceutical composition ofclaim 2, formulated for administration by subcutaneous injection. 16.The pharmaceutical composition of claim 2, comprising between 0.1 and200 mg of the compound per ml of the pharmaceutically-acceptablecarrier.
 17. The pharmaceutical composition of claim 2, comprisingbetween 0.1 and 100 mg of the compound per ml of thepharmaceutically-acceptable carrier.